Fusion protein comprising il13

ABSTRACT

The invention is concerned with a fusion protein comprising interleukin 13 and a regulatory cytokine, for example, an interleukin chosen from interleukin 4, interleukin 10, interleukin 27, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2, and interleukin 13, a nucleic acid molecule encoding such fusion protein, a vector comprising such nucleic acid molecule, and a host cell comprising such nucleic acid molecule or such vector. The invention further pertains to a method for producing such fusion protein. The fusion protein or a gene therapy vector encoding the fusion protein may be used in the prevention or treatment of a condition characterized by pathological pain, chronic pain, neuro-inflammation and/or or neurodegeneration.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No.17/158,785 filed Jan. 26, 2021, which is a continuation of InternationalApplication No. PCT/EP2020/060910, filed Apr. 17, 2020, which claims thebenefit of Dutch Patent Application No. 2022982, filed Apr. 19, 2019,and Dutch Patent Application No. 2022984, filed Apr. 19, 2019, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 22, 2021, isnamed 56780-702_401_SL.txt and is 67,224 bytes in size.

FIELD OF THE INVENTION

The present invention is in the field of neuro-immunology andpharmacology, particularly for treatment of chronic pain,neuro-inflammatory and neurodegenerative diseases, and inflammatorydisorders. The invention particularly relates to a novel fusion proteincomprising interleukin 13 (IL13) and a regulatory cytokine, for example,without limitation, an interleukin chosen from interleukin 4 (IL4),interleukin 10 (IL10), interleukin 27 (IL27), interleukin 33 (IL33),transforming growth factor beta 1 (TGFβ1), transforming growth factorbeta 2 (TGFβ2), and IL13 itself, either or not physically fused togetherthrough a linker sequence. Particularly, the present invention providesan IL4/IL13, IL10/IL13, IL27/IL13, IL33/IL13, TGFβ1/IL13, TGFβ2/IL13, orIL13/IL13 fusion protein endowed with a superior analgesic,neuro-protective, and anti-inflammatory activity over a combination ofthe individual cytokines, or over a fusion protein of IL4 and IL10. Thepresent invention also provides nucleic acid sequences encoding a fusionprotein, for example, an IL4/IL13 fusion protein, IL10/IL13 fusionprotein, IL27/IL13 fusion protein, IL33/IL13 fusion protein, TGFβ1/IL13fusion protein, TGFβ2/IL13 fusion protein, or IL13/IL13 fusion protein,expression vectors comprising such nucleic acid sequences, host cells orhost organisms altered to harbour the nucleic acid sequence encoding theIL4/IL13 fusion protein, IL10/IL13 fusion protein, IL27/IL13 fusionprotein, IL33/IL13 fusion protein, TGFβ1/IL13 fusion protein, TGFβ2/IL13fusion protein, IL/13/IL13 fusion protein, and the fusion proteinitself. The invention further provides methods for producing anIL4/IL13, IL10/IL13, IL27/IL13, IL33/IL13, TGFβ1/IL13, TGFβ2/IL13, orIL13/IL13 fusion protein using a cell or organism harbouring suchnucleic acid sequences. Transgenic organisms comprising the nucleic acidsequence of the invention are also provided. The present invention alsorelates to pharmaceutical compositions comprising for example, theIL4/IL13 or IL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13 orTGFβ2/IL13 or IL13/IL13 fusion protein. Finally, the use of the IL4/IL13or IL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 orIL13/IL13 fusion protein as a medicament, in particular for theprevention and/or treatment of chronic pain and of conditionscharacterized by neuro-inflammation, neuro-degeneration, or inflammationis taught herein.

BACKGROUND OF THE INVENTION

Chronic pain affects millions of people and constitutes the largestunmet need of modern medicine¹⁻⁴. In 2016, an estimated 20.4% of U.S.adults (50.0 million) had chronic pain and 8.0% of U.S. adults (19.6million) had high-impact chronic pain⁵. Opioids and non-steroidalanti-inflammatory drugs (NSAIDs) constitute the main classes of drugs tocombat pain. However, these analgesics (“pain-killers”) are oftenineffective and have severe side effects (addiction, gastrointestinalbleeding, cardiovascular, other). It is estimated that ˜50% of chronicpain patients (˜5-10% of the total population) do not receive adequatepain relief⁶.

People with chronic pain suffer from spontaneous pain, hyperalgesia (aheightened experience of pain to a noxious stimulus) and allodynia (paincaused by a normally non-painful stimulus). Pain has multiple causes andresults from biological processes at various anatomic levels⁷⁻¹⁰: thegeneration of stimuli that trigger sensory nerve endings in theperiphery; the stimulation, sensitization, and dysfunction of peripheralsensory neurons that transmit action potentials to the spinal cord;neurons and glial cells in the dorsal horn of the spinal cord whereaction potentials from peripheral neurons are transmitted to spinal painneurons via synapses; and finally central mechanisms in the brain. Thecontribution of all of these processes to different types of chronicpain varies. Depending on this contribution pain is discriminated inseveral types, including nociceptive pain, peripheral and centralneuropathic pain, and mixed types of pain. Analgesic drugs used in theclinic, target pain at only one level: NSAIDs reduce the generation ofnociceptive stimuli in the periphery, whereas opioids inhibit centralmechanisms. Together with their notorious toxic side effects, thisexplains the limited clinical efficacy of these analgesics.

Last decade it has become increasingly clear that pain signals are notstraightforwardly transferred to the brain but rather are modulated byneuro-inflammatory processes involving glial cells in the spinal cord aswell as sensory neurons. Cytokines are well-known to orchestrate immuneand inflammatory processes, and also are crucial in the control of pain.Pro-inflammatory cytokines enhance inflammation and promote pain whilstregulatory (e.g., anti-inflammatory) cytokines dampen inflammation. Thebalance between proinflammatory and regulatory cytokines determines theoutcome of inflammatory reactions in vivo¹¹. Although pain promotingeffects of pro-inflammatory cytokines are well-known¹², knowledge on therole of regulatory cytokines in pain is limited¹³. Blocking ofregulatory cytokines such as TGFβ, IL10, IL4, and IL13, severely impairsthe resolution of transient inflammatory hyperalgesia¹⁴ andchemotherapy-induced allodynia¹⁵, demonstrating a critical role forendogenous regulatory cytokines in pain resolution. This role ofcytokines goes far beyond reducing inflammation and is fundamentallydifferent from that of anti-inflammatory drugs such as corticosteroidswhich only have limited analgesic effects^(16,17). Notably, not onlyglial cells are modulated by cytokines, but also sensory neuronsthemselves can directly respond to cytokines. Indeed, sensory neuronsexpress receptors for all regulatory cytokines, though expressiondiffers among neuronal subsets¹⁸. Pain resulting from peripheralinflammation or nerve damage is associated with the activation of spinalmicroglia and astrocytes that promote pain by enhancing spinal painsignal transmission¹⁹⁻²².

Considering the role of neuro-inflammation in chronic pain, regulatorycytokines potentially can target pain at multiple levels. Indeed, theydampen stimulation of nociceptors by reducing inflammation, theysuppress sensitization and dysfunction of sensory neurons, and theyprevent activation of pain pathways in the spinal cord by attenuatingthe production of pro-inflammatory mediators by glial cells^(21,22).However, the analgesic effects of therapy with stand-alone regulatory(e.g., IL10 or IL13) or anti-inflammatory cytokines (IL1-receptorantagonist) are limited¹⁹, presumably because optimal analgesic activityrequires synergy of various regulatory cytokines, and because of theirrelatively poor bioavailability due to rapid clearance by the kidney.Therefore, a new strategy to resolve chronic pain with regulatorycytokines has been proposed using a fusion-protein of IL4 andIL10^(19,23). Intrathecal administration of analgesics is commonpractice in pain treatment as this reduces the dose and decreasestoxicity of an analgesic drug²⁴. Intrathecal injection of IL4/IL10fusion protein reduces pain in mouse models for a variety of differenttypes of pain¹⁹. Remarkably, three repeated intrathecal administrationsof IL4/IL10 fusion protein results in a sustained alleviation of pain(e.g., completely and permanently resolves chronic pain, such asnociceptive pain) induced by inflammation in the paw, without modulatingperipheral inflammation itself¹⁹. Interestingly, the efficacy ofIL4/IL10 fusion protein is superior to that of stand-alone wild-typecytokines, and even to that of the combination of these cytokines¹⁹.

Resolution of pain by IL4/IL10 fusion protein was also observed inneuropathic¹⁹ and osteoarthritis pain models²⁵. However, the effect ofIL4/IL10 fusion protein on neuropathic and osteoarthritis pain can betransient, even after multiple injections. Therefore, there is a needfor providing a molecule for prevention or treatment of neuropathic painand osteoarthritis pain that has a long-lasting analgesic effect.

SUMMARY

The present disclosure provides a single molecule that targetsneuro-inflammation and -degeneration and has a long-lasting effect onchronic neuropathic pain. This molecule can be used for the treatment ofvarious diseases or disorders with different etiology, in which chronicpain, neuro-inflammation and/or neuro-degeneration play a role. Thepresent disclosure provides fusion proteins that comprise an interleukin13 (IL13) directly or indirectly linked to an a regulatory cytokine.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, for use in treatment of neuropathy in a subject inneed thereof.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, for use in treatment of pain in a subject in needthereof.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, for use in treatment of neurodegeneration orneuroinflammation in a subject in need thereof.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, for use in treatment of inflammation in a subjectin need thereof.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, for use in promoting neuroprotection in a subjectin need thereof.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, for use in modulating activity of a signalingpathway in a nervous system cell.

In some embodiments, the regulatory cytokine is selected from the groupconsisting of an interleukin 4 (IL4), an interleukin 10 (IL10), aninterleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), atransforming growth factor beta 2 (TGFβ2), and an additional interleukin13 (IL13). In some embodiments, the regulatory cytokine is IL4. In someembodiments, the regulatory cytokine is IL10. In some embodiments, theregulatory cytokine is IL33. In some embodiments, the regulatorycytokine is an interleukin 27 (IL27). In some embodiments, theregulatory cytokine is TGFβ1. In some embodiments, the regulatorycytokine is TGFβ2. In some embodiments, the regulatory cytokine is anadditional IL13. In some embodiments, the IL13 comprises a wild typeIL13. In some embodiments, the IL13 is a mammalian IL13. In someembodiments, the IL13 is a human IL13. In some embodiments, theregulatory cytokine comprises a wild type regulatory cytokine. In someembodiments, the regulatory cytokine is a mammalian regulatory cytokine.In some embodiments, the regulatory cytokine is a human regulatorycytokine. In some embodiments, the interleukin 27 comprises aninterleukin 27 alpha (IL27A). In some embodiments, the IL27A comprisesan L134C substitution relative to SEQ ID NO: 36. In some embodiments,the IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with anaffinity that is less than two fold increased and less than two folddecreased compared to a wild type IL13. In some embodiments, the IL13binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with an affinitythat is less than two fold increased and less than two fold decreasedcompared to a wild type IL13. In some embodiments, the IL13 binds to aninterleukin 4 receptor alpha (IL-4Rα) with an affinity that is less thantwo fold increased and less than two fold decreased compared to a wildtype IL13. In some embodiments, the regulatory cytokine amino acidsequence is a derivative sequence that binds to all subunits of areceptor of the regulatory cytokine with a comparable affinity as a wildtype regulatory cytokine. In some embodiments, the regulatory cytokineamino acid sequence is a derivative sequence that activates a nativereceptor of the regulatory cytokine. In some embodiments, the IL13comprises an amino acid sequence with at least 90% sequence identity toa sequence selected from the group consisting of SEQ ID NO: 2 and anyone of SEQ ID NOs: 9-15. In some embodiments, the IL13 comprises anamino acid sequence that is selected from the group consisting of SEQ IDNO: 2 and any one of SEQ ID NOs: 9-15. In some embodiments, the IL13comprises an amino acid sequence with between 1 and 10 amino aciddeletions, insertions, substitutions, or a combination thereof relativeto a sequence selected from the group consisting of SEQ ID NO: 2 and anyone of SEQ ID NOs: 9-15. In some embodiments, the regulatory cytokinecomprises an amino acid sequence with at least 90% sequence identity toa sequence selected from the group consisting of SEQ ID NO: 1, any oneof SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO:29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ IDNo: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO: 45. In someembodiments, the regulatory cytokine comprises an amino acid sequencethat is selected from the group consisting of SEQ ID NO: 1, any one ofSEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO:29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ IDNo: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO: 45. In someembodiments, the regulatory cytokine comprises an amino acid sequencewith between 1 and 10 amino acid deletions, insertions, substitutions,or a combination thereof relative to a sequence selected from the groupconsisting SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQ ID NO: 36,and SEQ ID NO: 45. In some embodiments, the IL13 and the regulatorycytokine are covalently linked. In some embodiments, the IL13 and theregulatory cytokine are joined by a linker. In some embodiments, a Cterminus of the IL13 is joined to an N-terminus of the cytokine,optionally via a linker. In some embodiments, an N terminus of the IL13is joined to a C-terminus of the cytokine, optionally via a linker. Insome embodiments, the fusion protein further comprises one or morechemical modifications. In some embodiments, the one or more chemicalmodifications are selected from the group consisting of glycosylation,fucosylation, sialylation, and pegylation. In some embodiments, theprotein construct comprises an affinity tag. In some embodiments, theneuropathy is post-traumatic peripheral neuropathy, post-operativeperipheral neuropathy, diabetic peripheral neuropathy, inflammatoryperipheral neuropathy, HIV-associated neuropathy, chemotherapy-inducedneuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy,cranial neuropathy, predominantly motor neuropathy, predominantlysensory neuropathy, sensory-motor neuropathy, autonomic neuropathy,idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia,glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia,atypical trigeminal neuralgia, sciatica, brachial plexopathy, orintercostal neuralgia. In some embodiments, the neuropathy is associatedwith pain, numbness, weakness, burning, atrophy, tingling, twitching, ora combination thereof. In some embodiments, the pain is chronic pain. Insome embodiments, the pain is pathological pain, inflammatory pain,neuropathic pain, nociceptive pain, or mixed nociceptive-neuropathicpain. In some embodiments, the pain is visceral nociceptive pain,non-visceral nociceptive pain, peripheral neuropathic pain, centralneuropathic pain, or a combination thereof. In some embodiments, thepain is post-operative orthopedic surgery pain, musculoskeletal pain,chemotherapy-associated pain, chemotherapy-induced allodynia,post-spinal cord injury pain, post-stroke pain, low back pain, cancerpain, or chronic visceral pain. In some embodiments, the pain isassociated with irritable bowel syndrome, inflammatory bowel disease,rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia,trigeminal neuralgia, post-traumatic peripheral neuropathy,post-operative peripheral neuropathy, diabetic peripheral neuropathy,inflammatory peripheral neuropathy, HIV-associated neuropathy,peripheral neuropathy, nerve entrapment syndrome, chemotherapy-inducedneuropathy, multiple sclerosis, chemotherapy-induced neurodegeneration,complex regional pain syndrome, osteoarthritis, fibromyalgia,polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease,Parkinson's disease, Huntington's disease, polyneuropathy, oramyotrophic lateral sclerosis. In some embodiments, the pain isassociated with Alpers' Disease, Arachnoiditis, Arthrofibrosis, AtaxicCerebral Palsy, Autoimmune Atrophic Gastritis, Amyloidosis, hATTRAmyloidosis, Avascular Necrosis, Back Pain, Batten Disease, Behçet'sDisease (Syndrome), Breakthrough Pain, Burning Mouth Syndrome, Bursitis,Central Autosomal Dominant Arteriopathy with Subcortical Infarcts andLeukoencephalopathy (Cadasil), Cerebral ischemia,Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnel syndrome,Cauda Equina Syndrome, Central Pain Syndrome, Cerebral Palsy,Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis, Charcot-Marie-Tooth(CMT) Disease, Chronic Functional Abdominal Pain (CFAP), ChronicPancreatitis, Collapsed Lung (Pneumothorax), Corticobasal Degeneration,Compression injury, Corneal Neuropathic Pain, Crush syndrome,Degenerative Disc Disease, Dermatomyositis, Dementia, Dystonia,Ehlers-Danlos Syndrome (EDS), Endometriosis, Eosinophilia-MyalgiaSyndrome (EMS), Erythromelalgia, Failed Back Surgery Syndrome (FBSS),Fibromyalgia, Friedreich's Ataxia, Frontotemporal dementia,Glossopharyngeal neuralgia, Growing Pains, Herniated disc,Hydrocephalus, Intercostal Neuraligia, Interstitial Cystitis, JuvenileDermatositis, Knee Injury, Leg Pain, Lewy Body Dementia, LoinPain-Haematuria Syndrome, Lyme Disease, Meralgia Paresthetica,Mitochondrial Disorders, Mixed dementia, Motor neurone diseases (MND),Monomelic Amyotrophy, Multiple system atrophy (MSA), Myositis, NeckPain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis, Paget'sDisease, Parsonage Turner Syndrome, Pelvic Pain, Peripheral Neuropathy,Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis, PolymyalgiaRhuematica, Polymyositis, Post Herniorraphy Pain Syndrome, PostMastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy PainSyndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, PsoriaticArthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome,Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis,Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), Herpes Zoster Shingles, SpasmodicTorticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, SpinalStenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, ThoracicOutlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, TraumaticBrain Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combinationthereof. In some embodiments, the neurodegeneration or neuroinflammationcomprises Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, multiple sclerosis,spinocerebellar ataxia, or spinal muscular atrophy. In some embodiments,the inflammation comprises chronic inflammation. In some embodiments,the inflammation comprises local inflammation or systemic inflammation.In some embodiments, the inflammation is associated with inflammatorybowel disease, irritable bowel syndrome, osteoarthritis, rheumatoidarthritis, glomerulonephritis, sepsis, adult respiratory distresssyndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis,ankylosing spondylarthritis, systemic lupus erythematosus, vasculitis,gout, allotransplantation, xenotransplantation, an autoimmune disease,Sjogren's disease, a burn injury, trauma, stroke, myocardial infarction,atherosclerosis, diabetes mellitus, extracorporeal dialysis and bloodoxygenation, ischemia-reperfusion injuries, and toxicity induced by thein vivo administration of cytokines or other therapeutic monoclonalantibodies. In some embodiments, nerve fiber degeneration is reduced. Insome embodiments, nerve fiber loss is reduced. In some embodiments,maintenance of nerve fiber density is promoted. In some embodiments,nerve fiber regrowth is promoted. In some embodiments, neuroprotectionin the central nervous system is promoted. In some embodiments,neuroprotection in the peripheral nervous system is promoted. In someembodiments, intraepidermal nerve fiber loss is reduced. In someembodiments, the neuronal dysfunction is reduced. In some embodiments,the fusion protein elicits a therapeutic effect of greater magnitudethan equivalent amounts of the IL13, the regulatory cytokine, or acombination thereof. In some embodiments, the fusion protein elicits atherapeutic effect of greater duration than equivalent amounts of theIL13, the regulatory cytokine, or a combination thereof. In someembodiments, the fusion protein is present in a pharmaceuticalcomposition comprising the fusion protein and one or morepharmaceutically-acceptable excipients. In some embodiments, thepharmaceutical composition is in a unit dosage form. In someembodiments, the fusion protein is present in the pharmaceuticalcomposition at a concentration of about 50 μg per mL to about 100 mg permL. In some embodiments, the fusion protein is formulated foradministration in a dose of between about 0.5 μg per kg of body weightto about 1 mg per kg of body weight. In some embodiments, the fusionprotein is formulated for administration in a controlled releaseformulation. In some embodiments, the fusion protein is formulated foradministration by a parenteral, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intrasternal, intracerebral, intraocular, intralesional,intracerebroventricular, intracisternal, or intraparenchymal route. Insome embodiments, the nervous system cell is a neuron. In someembodiments, the nervous system cell is a central nervous system cell.In some embodiments, the nervous system cell is a peripheral nervoussystem cell. In some embodiments, the neuron is a sensory neuron. Insome embodiments, the neuron is a somatosensory neuron. In someembodiments, the neuron is a visceral sensory neuron. In someembodiments, the neuron is a nociceptor. In some embodiments, the neuronis an autonomic neuron. In some embodiments, the nervous system cell isa glial cell. In some embodiments, the nervous system cell is amicroglial cell. In some embodiments, the nervous system cell is aninfiltrating cell. In some embodiments, the nervous system cell is aninfiltrating macrophage. In some embodiments, the signaling pathway ismodulated in a presence of a pro-inflammatory mediator.

Disclosed herein, in some aspects, is a method of treating neuropathy ina subject in need thereof, comprising administering to the subject aneffective amount of a fusion protein that comprises an interleukin 13(IL13) amino acid sequence and a regulatory cytokine amino acidsequence.

Disclosed herein, in some aspects, is a method of treating pain in asubject in need thereof, comprising administering to the subject aneffective amount of a fusion protein that comprises an interleukin 13(IL13) amino acid sequence and a regulatory cytokine amino acidsequence.

Disclosed herein, in some aspects, is a method of treatingneurodegeneration or neuroinflammation in a subject in need thereof,comprising administering to the subject an effective amount of a fusionprotein that comprises an interleukin 13 (IL13) amino acid sequence anda regulatory cytokine amino acid sequence.

Disclosed herein, in some aspects, is a method of treating inflammationin a subject in need thereof, comprising administering to the subject aneffective amount of a fusion protein that comprises an interleukin 13(IL13) amino acid sequence and a regulatory cytokine amino acidsequence.

Disclosed herein, in some aspects, is a method of promotingneuroprotection in a subject in need thereof, comprising administeringto the subject an effective amount of a fusion protein that comprises aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence.

Disclosed herein, in some aspects, is a method of modulating activity ofa signaling pathway in nervous system cell, comprising contacting thenervous system cell with a fusion protein that comprises an interleukin13 (IL13) amino acid sequence and a regulatory cytokine amino acidsequence.

In some embodiments, the regulatory cytokine is selected from the groupconsisting of an interleukin 4 (IL4), an interleukin 10 (IL10), aninterleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), atransforming growth factor beta 2 (TGFβ2), and an additional interleukin13 (aIL13). In some embodiments, the regulatory cytokine is IL4. In someembodiments, the regulatory cytokine is IL10. In some embodiments, theregulatory cytokine is IL33. In some embodiments, the regulatorycytokine is an interleukin 27 (IL27). In some embodiments, theregulatory cytokine is TGFβ1. In some embodiments, the regulatorycytokine is TGFβ2. In some embodiments, the regulatory cytokine is anaIL13. In some embodiments, the IL13 comprises a wild type IL13. In someembodiments, the IL13 is a mammalian IL13. In some embodiments, the IL13is a human IL13. In some embodiments, the regulatory cytokine comprisesa wild type regulatory cytokine. In some embodiments, the regulatorycytokine is a mammalian regulatory cytokine. In some embodiments, theregulatory cytokine is a human regulatory cytokine. In some embodiments,the interleukin 27 comprises an interleukin 27 alpha (IL27A). In someembodiments, the IL27A comprises an L134C substitution relative to SEQID NO: 36. In some embodiments, the IL13 binds to interleukin 13receptor alpha 1 (IL-13Rα1) with an affinity that is less than two foldincreased and less than two fold decreased compared to a wild type IL13.In some embodiments, the IL13 binds to interleukin 13 receptor alpha 2(IL-13Rα2) with an affinity that is less than two fold increased andless than two fold decreased compared to a wild type IL13. In someembodiments, the IL13 binds to an interleukin 4 receptor alpha (IL-4Rα)with an affinity that is less than two fold increased and less than twofold decreased compared to a wild type IL13. In some embodiments, theregulatory cytokine amino acid sequence is a derivative sequence thatbinds to all subunits of a receptor of the regulatory cytokine with acomparable affinity as a wild type regulatory cytokine. In someembodiments, the regulatory cytokine amino acid sequence is a derivativesequence that activates a native receptor of the regulatory cytokine. Insome embodiments, the IL13 comprises an amino acid sequence with atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15. In someembodiments, the IL13 comprises an amino acid sequence that is selectedfrom the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs:9-15. In some embodiments, the IL13 comprises an amino acid sequencewith between 1 and 10 amino acid deletions, insertions, substitutions,or a combination thereof relative to a sequence selected from the groupconsisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15. In someembodiments, the regulatory cytokine comprises an amino acid sequencewith at least 90% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ IDNO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ IDNO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQID NO: 36, and SEQ ID NO: 45. In some embodiments, the regulatorycytokine comprises an amino acid sequence that is selected from thegroup consisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ IDNO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ IDNO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQID NO: 36, and SEQ ID NO: 45. In some embodiments, the regulatorycytokine comprises an amino acid sequence with between 1 and 10 aminoacid deletions, insertions, substitutions, or a combination thereofrelative to a sequence selected from the group consisting SEQ ID NO: 1,any one of SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQID NO: 29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22,SEQ ID No: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO: 45. In someembodiments, the IL13 and the regulatory cytokine are covalently linked.In some embodiments, the IL13 and the regulatory cytokine are joined bya linker. In some embodiments, a C terminus of the IL13 is joined to anN-terminus of the cytokine, optionally via a linker. In someembodiments, an N terminus of the IL13 is joined to a C-terminus of thecytokine, optionally via a linker. In some embodiments, the fusionprotein further comprises one or more chemical modifications. In someembodiments, the one or more chemical modifications are selected fromthe group consisting of glycosylation, fucosylation, sialylation, andpegylation. In some embodiments, the protein construct comprises anaffinity tag. In some embodiments, the neuropathy is post-traumaticperipheral neuropathy, post-operative peripheral neuropathy, diabeticperipheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, chemotherapy-induced neuropathy,polyneuropathy, mononeuropathy, multiple mononeuropathy, cranialneuropathy, predominantly motor neuropathy, predominantly sensoryneuropathy, sensory-motor neuropathy, autonomic neuropathy, idiopathicneuropathy, post-herpetic neuralgia, trigeminal neuralgia,glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia,atypical trigeminal neuralgia, sciatica, brachial plexopathy, orintercostal neuralgia. In some embodiments, the neuropathy is associatedwith pain, numbness, weakness, burning, atrophy, tingling, twitching, ora combination thereof. In some embodiments, the pain is chronic pain. Insome embodiments, the pain is neuropathic pain, nociceptive pain, ormixed nociceptive-neuropathic pain. In some embodiments, the pain isvisceral nociceptive pain, non-visceral nociceptive pain, peripheralneuropathic pain, central neuropathic pain, or a combination thereof. Insome embodiments, the pain is post-operative orthopedic surgery pain,musculoskeletal pain, chemotherapy-associated pain, chemotherapy-inducedallodynia, post-spinal cord injury pain, post-stroke pain, low backpain, cancer pain, or chronic visceral pain. In some embodiments, thepain is associated with irritable bowel syndrome, inflammatory boweldisease, rheumatoid arthritis, ankylosing spondylitis, post-herpeticneuralgia, trigeminal neuralgia, post-traumatic peripheral neuropathy,post-operative peripheral neuropathy, diabetic peripheral neuropathy,inflammatory peripheral neuropathy, HIV-associated neuropathy,peripheral neuropathy, nerve entrapment syndrome, chemotherapy-inducedneuropathy, multiple sclerosis, chemotherapy-induced neurodegeneration,complex regional pain syndrome, osteoarthritis, fibromyalgia,polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease,Parkinson's disease, Huntington's disease, polyneuropathy, oramyotrophic lateral sclerosis. In some embodiments, the pain isassociated with Alpers' Disease, Arachnoiditis, Arthrofibrosis, AtaxicCerebral Palsy, Autoimmune Atrophic Gastritis, Amyloidosis, hATTRAmyloidosis, Avascular Necrosis, Back Pain, Batten Disease, Behçet'sDisease (Syndrome), Breakthrough Pain, Burning Mouth Syndrome, Bursitis,Central Autosomal Dominant Arteriopathy with Subcortical Infarcts andLeukoencephalopathy (Cadasil), Cerebral ischemia,Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnel syndrome,Cauda Equina Syndrome, Central Pain Syndrome, Cerebral Palsy,Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis, Charcot-Marie-Tooth(CMT) Disease, Chronic Functional Abdominal Pain (CFAP), ChronicPancreatitis, Collapsed Lung (Pneumothorax), Corticobasal Degeneration,Compression injury, Corneal Neuropathic Pain, Crush syndrome,Degenerative Disc Disease, Dermatomyositis, Dementia, Dystonia,Ehlers-Danlos Syndrome (EDS), Endometriosis, Eosinophilia-MyalgiaSyndrome (EMS), Erythromelalgia, Failed Back Surgery Syndrome (FBSS),Fibromyalgia, Friedreich's Ataxia, Frontotemporal dementia,Glossopharyngeal neuralgia, Growing Pains, Herniated disc,Hydrocephalus, Intercostal Neuraligia, Interstitial Cystitis, JuvenileDermatositis, Knee Injury, Leg Pain, Lewy Body Dementia, LoinPain-Haematuria Syndrome, Lyme Disease, Meralgia Paresthetica,Mitochondrial Disorders, Mixed dementia, Motor neurone diseases (MND),Monomelic Amyotrophy, Multiple system atrophy (MSA), Myositis, NeckPain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis, Paget'sDisease, Parsonage Turner Syndrome, Pelvic Pain, Peripheral Neuropathy,Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis, PolymyalgiaRhuematica, Polymyositis, Post Herniorraphy Pain Syndrome, PostMastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy PainSyndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, PsoriaticArthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome,Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis,Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), Herpes Zoster Shingles, SpasmodicTorticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, SpinalStenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, ThoracicOutlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, TraumaticBrain Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combinationthereof. In some embodiments, the neurodegeneration or neuroinflammationcomprises Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, multiple sclerosis,spinocerebellar ataxia, or spinal muscular atrophy. In some embodiments,the inflammation comprises chronic inflammation. In some embodiments,the inflammation comprises local inflammation or systemic inflammation.In some embodiments, the inflammation is associated with inflammatorybowel disease, irritable bowel syndrome, osteoarthritis, rheumatoidarthritis, glomerulonephritis, sepsis, adult respiratory distresssyndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis,ankylosing spondylarthritis, systemic lupus erythematosus, vasculitis,gout, allotransplantation, xenotransplantation, an autoimmune disease,Sjogren's disease, a burn injury, trauma, stroke, myocardial infarction,atherosclerosis, diabetes mellitus, extracorporeal dialysis and bloodoxygenation, ischemia-reperfusion injuries, and toxicity induced by thein vivo administration of cytokines or other therapeutic monoclonalantibodies. In some embodiments, IL13 treatment is indicated. In someembodiments, IL4, IL10, IL27, IL33, TGFβ1, or TGFβ2 treatment isindicated. In some embodiments, the method reduces nerve fiberdegeneration. In some embodiments, the method reduces nerve fiber loss.In some embodiments, the method promotes maintenance of nerve fiberdensity. In some embodiments, the method promotes nerve fiber regrowth.In some embodiments, the method promotes neuroprotection in the centralnervous system. In some embodiments, the method promotes neuroprotectionin the peripheral nervous system. In some embodiments, the methodreduces intraepidermal nerve fiber loss. In some embodiments, the methodreduces neuronal dysfunction. In some embodiments, administering thefusion protein elicits a therapeutic effect of greater magnitude thanadministering equivalent amounts of the IL13, the regulatory cytokine,or a combination thereof. In some embodiments, administering the fusionprotein elicits a therapeutic effect of greater duration thanadministering equivalent amounts of the IL13, the regulatory cytokine,or a combination thereof. In some embodiments, administering results ina higher magnitude of pain alleviation as compared to a comparableamount of IL13 and the regulatory cytokine administered individually orin combination as measured by mechanical sensitivity to von Frey hairsin a paclitaxel-induced mouse model of neuropathy. In some embodiments,the fusion protein is present in a pharmaceutical composition comprisingthe fusion protein and one or more pharmaceutically-acceptableexcipients. In some embodiments, the composition is in a unit dosageform. In some embodiments, the fusion protein is present in thepharmaceutical composition at a concentration of about 50 μg to about100 mg per mL. In some embodiments, the fusion protein is administeredin a dose of between about 0.5 μg to 1 mg per kg of body weight. In someembodiments, the fusion protein is administered in a controlled releaseformulation. In some embodiments, the fusion protein is administered bya parenteral, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural, intrasternal, intracerebral,intraocular, intralesional, intracerebroventricular, intracisternal, orintraparenchymal route. In some embodiments, the nervous system cell isa neuron. In some embodiments, the nervous system cell is a centralnervous system cell. In some embodiments, the nervous system cell is aperipheral nervous system cell. In some embodiments, the neuron is asensory neuron. In some embodiments, the neuron is a somatosensoryneuron. In some embodiments, the neuron is a visceral sensory neuron. Insome embodiments, the neuron is a nociceptor. In some embodiments, theneuron is an autonomic neuron. In some embodiments, the nervous systemcell is a glial cell. In some embodiments, the nervous system cell is amicroglial cell. In some embodiments, the nervous system cell is aninfiltrating cell. In some embodiments, the nervous system cell is aninfiltrating macrophage. In some embodiments, the signaling pathway ismodulated in a presence of a pro-inflammatory mediator.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence that is a wild type IL13sequence and a regulatory cytokine amino acid sequence.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence that is a wild type sequence.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, wherein the IL13 amino acid sequence is an IL13derivative sequence that binds to interleukin 13 receptor alpha 1(IL-13Rα1), interleukin 13 receptor alpha 2 (IL-13Rα2), and interleukin4 receptor alpha (IL-4Rα) with a comparable affinity as a wild typeinterleukin 13 sequence.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence, wherein the regulatory cytokine amino acid sequenceis a derivative sequence that binds to all receptor subunits that a wildtype version of the regulatory cytokine binds with a comparable affinityas the wild type regulatory cytokine.

In some embodiments, the regulatory cytokine is selected from the groupconsisting of an interleukin 4 (IL4), an interleukin 10 (IL10), aninterleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), atransforming growth factor beta 2 (TGFβ2), and an additional interleukin13 (aIL13). In some embodiments, the regulatory cytokine is IL4. In someembodiments, the regulatory cytokine is IL10. In some embodiments, theregulatory cytokine is IL33. In some embodiments, the regulatorycytokine is an interleukin 27 (IL27). In some embodiments, theregulatory cytokine is TGFβ1. In some embodiments, the regulatorycytokine is TGFβ2. In some embodiments, the regulatory cytokine is anadditional interleukin 13 (aIL13). In some embodiments, the IL13 is amammalian IL13. In some embodiments, the IL13 is a human IL13. In someembodiments, the IL13 comprises a wild type IL13. In some embodiments,the regulatory cytokine comprises a wild type regulatory cytokine. Insome embodiments, the regulatory cytokine is a mammalian regulatorycytokine. In some embodiments, the regulatory cytokine is a humanregulatory cytokine. In some embodiments, the interleukin 27 comprisesan interleukin 27 alpha (IL27A). In some embodiments, the IL27Acomprises an L134C substitution relative to SEQ ID NO: 36. In someembodiments, the IL13 binds to interleukin 13 receptor alpha 1(IL-13Rα1) with an affinity that is less than two fold increased andless than two fold decreased compared to a wild type IL13. In someembodiments, the IL13 binds to interleukin 13 receptor alpha 2(IL-13Rα2) with an affinity that is less than two fold increased andless than two fold decreased compared to a wild type IL13. In someembodiments, the IL13 binds to an interleukin 4 receptor alpha (IL-4Rα)with an affinity that is less than two fold increased and less than twofold decreased compared to a wild type IL13. In some embodiments, theregulatory cytokine amino acid sequence is a derivative sequence thatbinds to all subunits of a receptor of the regulatory cytokine with acomparable affinity as a wild type regulatory cytokine. In someembodiments, the regulatory cytokine amino acid sequence is a derivativesequence that activates a native receptor of the regulatory cytokine. Insome embodiments, the IL13 comprises an amino acid sequence with atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15. In someembodiments, the IL13 comprises an amino acid sequence that is selectedfrom the group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs:9-15. In some embodiments, the IL13 comprises an amino acid sequencewith between 1 and 10 amino acid deletions, insertions, substitutions,or a combination thereof relative to a sequence selected from the groupconsisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15. In someembodiments, the regulatory cytokine comprises an amino acid sequencewith at least 90% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ IDNO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ IDNO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQID NO: 36, and SEQ ID NO: 45. In some embodiments, the regulatorycytokine comprises an amino acid sequence that is selected from thegroup consisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ IDNO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ IDNO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQID NO: 36, and SEQ ID NO: 45. In some embodiments, the regulatorycytokine comprises an amino acid sequence with between 1 and 10 aminoacid deletions, insertions, substitutions, or a combination thereofrelative to a sequence selected from the group consisting SEQ ID NO: 1,any one of SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQID NO: 29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22,SEQ ID NO: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO: 45. In someembodiments, the IL13 and the regulatory cytokine are covalently linked.In some embodiments, the IL13 and the regulatory cytokine are joined bya linker. In some embodiments, a C terminus of the IL13 is joined to anN-terminus of the cytokine, optionally via a linker. In someembodiments, an N terminus of the IL13 is joined to a C-terminus of thecytokine, optionally via a linker. In some embodiments, the fusionprotein further comprises one or more chemical modifications. In someembodiments, the one or more chemical modifications are selected fromthe group consisting of glycosylation, fucosylation, sialylation, andpegylation. In some embodiments, the protein construct comprises anaffinity tag. In some embodiments, a nucleic acid molecule is providedthat encodes the fusion protein. In some embodiments, the nucleic acidmolecule is codon optimized for expression in the cell. In someembodiments, the nucleic acid molecule is a vector. In some embodiments,a cell comprises the nucleic acid. In some embodiments, the fusionprotein is present in a pharmaceutical composition that also comprises apharmaceutically-acceptable excipient. In some embodiments, the nucleicacid vectors is present in a pharmaceutical composition that alsocomprises a pharmaceutically-acceptable excipient. In some embodiments,the pharmaceutical composition is in a unit dosage form. In someembodiments, the fusion protein is present in the pharmaceuticalcomposition at about 50 μg to about 100 mg per mL. In some embodiments,the fusion protein is formulated for administration as a dose of betweenabout 0.5 μg to 1 mg per kg of body weight. In some embodiments, thefusion protein formulated for administration as a controlled releaseformulation. In some embodiments, the pharmaceutical composition isformulated for administration by a parenteral, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural, intrasternal, intracerebral, intraocular, intralesional,intracerebroventricular, intracisternal, or intraparenchymal route. Insome embodiments, an effective amount of the pharmaceutical compositionis administered to a subject in need thereof. In some embodiments, thefusion protein is produced by comprising culturing a cell underconditions that permit the production of the fusion protein, wherein thecell comprises the polynucleotide sequence. In some embodiments, thefusion protein is harvested. In some embodiments, the fusion protein ispurified from harvested culture medium. In some embodiments, the fusionprotein is used for crosslinking an interleukin 13 receptor with aregulatory cytokine receptor.

Disclosed herein, in some aspects, is a fusion protein comprising aninterleukin 13 and an interleukin chosen from interleukin 4, interleukin10, interleukin 33, transforming growth factor beta 1, transforminggrowth factor beta 2, and interleukin 13.

In some embodiments, the interleukin 13 and said interleukin chosen frominterleukin 4, interleukin 10, interleukin 33, transforming growthfactor beta 1, transforming growth factor beta 2, and interleukin 13,are linked by a linker sequence. In some embodiments, the interleukin 13is fused N-terminal of the interleukin chosen from interleukin 4,interleukin 10, interleukin 33, transforming growth factor beta 1,transforming growth factor beta 2, and interleukin 13. In someembodiments, the interleukin chosen from interleukin 4, interleukin 10,interleukin 33, transforming growth factor beta 1, transforming growthfactor beta 2, and interleukin 13, is fused N-terminal of theinterleukin 13. In some embodiments, the fusion protein furthercomprises one or more chemical modification(s). In some embodiments, thechemical modification is selected from the group consisting ofglycosylation, fucosylation, sialylation, and pegylation. In someembodiments, the interleukin 13 is human interleukin 13. In someembodiments, the interleukin 4 is human interleukin 4, and/or saidinterleukin 10 is human interleukin 10, and/or said interleukin 33 ishuman interleukin 33, and/or said transforming growth factor beta 1 ishuman transforming growth factor beta 1, and/or said transforming growthfactor beta 2 is human transforming growth factor beta 2. In someembodiments, the fusion protein is encoded by a polynucleotide presentin a nucleic acid molecule. In some embodiments, the nucleic acidmolecule is present in a vector. In some embodiments, the nucleic acidmolecule is present in a host cell. In some embodiments, the fusionprotein is made by a method comprising the steps of: culturing the hostcell under conditions permitting the production of the fusion protein,optionally, purifying the fusion protein from the conditioned culturemedium. In some embodiments, the fusion protein is present in apharmaceutical composition that also comprises a pharmaceuticallyacceptable carrier. In some embodiments, the fusion protein is used as amedicament. In some embodiments, the fusion protein is used in theprevention or treatment of a condition characterized by chronic pain,neuro-inflammation or neuro-degeneration. In some embodiments, thecondition is further characterized by visceral or non-visceralnociceptive pain, peripheral or central neuropathic pain, or mixednociceptive-neuropathic pain, neuro-inflammation, and/orneuro-degeneration. In some embodiments, the condition is selected fromthe group consisting of post-operative orthopedic surgery pain,musculoskeletal pain, irritable bowel syndrome, inflammatory boweldisease, rheumatoid arthritis, ankylosing spondylitis, post-herpeticneuralgia, trigeminal neuralgia, post-traumatic or post-operativeperipheral neuropathy, diabetic peripheral neuropathy, inflammatoryperipheral neuropathy, HIV-associated neuropathy, painful peripheralneuropathy, nerve entrapment syndrome, chemotherapy-associated pain,chemotherapy-induced allodynia, complex regional pain syndrome,post-spinal injury pain, post-stroke pain, multiple sclerosis, low backpain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia,polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's diseaseand Parkinson's disease, Huntington's disease, and/or amyotrophiclateral sclerosis, or multiple sclerosis. In some embodiments, thefusion protein is used in the prevention or treatment of a clinicalcondition in a mammal, such as a human, for which interleukin 13 isindicated. In some embodiments, the fusion protein is used in theprevention or treatment of a clinical condition in a mammal, such as ahuman, for which interleukin 4 and/or interleukin 10 and/or interleukin33 and/or interleukin 27 and/or transforming growth factor beta 1 and/ortransforming growth factor beta 2, is indicated.

Disclosed herein, in some aspects is a gene therapy vector containingnucleotide sequence(s) coding for interleukin 13 and an interleukinchosen from interleukin 4, interleukin 10, interleukin 27, interleukin33, transforming growth factor beta 1, transforming growth factor beta2, and interleukin 13, for use in the prevention or treatment of acondition characterized by chronic pain, neuro-inflammation and/orneuro-degeneration.

In some embodiments, the condition is further characterized by visceralor non-visceral nociceptive pain, peripheral or central neuropathicpain, or mixed nociceptive-neuropathic pain, neuro-inflammation, and/orneuro-degeneration. In some embodiments, the condition is selected fromthe group consisting of post-operative orthopedic surgery pain,musculoskeletal pain, irritable bowel syndrome, inflammatory boweldisease, rheumatoid arthritis, ankylosing spondylitis, post-herpeticneuralgia, trigeminal neuralgia, post-traumatic or post-operativeperipheral neuropathy, diabetic peripheral neuropathy, inflammatoryperipheral neuropathy, HIV-associated neuropathy, painful peripheralneuropathy, nerve entrapment syndrome, chemotherapy-associated pain,chemotherapy-induced allodynia, complex regional pain syndrome,post-spinal injury pain, post-stroke pain, multiple sclerosis, low backpain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia,polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's diseaseand Parkinson's disease, Huntington's disease, and/or amyotrophiclateral sclerosis, or multiple sclerosis.

In a some aspects, the present invention relates to a fusion proteincomprising at least 2, 3, 4, preferably 2 regulatory (e.g.,anti-inflammatory) interleukins chosen from the group consisting ofinterleukin 13 (IL13), interleukin 4 (IL4), interleukin 10 (IL10),interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factorbeta 1 (TGFβ1), and transforming growth factor beta 2 (TGFβ2).

Preferably, the present invention relates to a fusion protein comprisingan IL13 and another, i.e. a second, interleukin/cytokine, preferablychosen from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, and IL13 itself.

In an embodiment, said IL13 and said interleukin chosen from IL4, IL10,IL27, IL33, TGFβ1, TGFβ2, or IL13 itself are connected by a linker.

In an embodiment, the interleukin chosen from IL4, IL10, IL27, IL33,TGFβ1, TGFβ2, or IL13 itself is fused N-terminal of the IL13.

In another embodiment, the IL13 is fused N-terminal of the interleukinchosen from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or IL13 itself.

In an embodiment, said fusion protein further comprises one or morechemical modifications. Said chemical modifications may be selected fromthe group consisting of glycosylation, fucosylation, sialylation, andpegylation.

In an embodiment, said IL13 is human IL13.

In an embodiment said IL4 is human IL4.

In an embodiment, said IL10 is human IL10.

In an embodiment, said IL27 is human IL27.

In an embodiment, said IL33 is human IL33.

In an embodiment, said TGFβ1 is human TGFβ1.

In an embodiment, said TGFβ2 is human TGFβ2

In a second aspect, the present invention pertains to a nucleic acidmolecule comprising a polynucleotide encoding the fusion protein taughtherein.

In another aspect, the present invention is directed to a vectorcomprising the nucleic acid molecule taught herein.

In an aspect, the present invention is concerned with a host cellcomprising the nucleic acid molecule taught herein or the vector taughtherein.

In an aspect, the present invention provides a method for producing afusion protein as taught herein, said method comprising the steps of:culturing a host cell as taught herein under conditions permitting theproduction of the fusion protein as taught herein; and optionally,recovering the fusion protein.

In yet another aspect, the present invention provides for apharmaceutical composition comprising the fusion protein as taughtherein, and a pharmaceutically acceptable carrier or excipient.

The invention also pertains to a fusion protein as taught herein for useas a medicament, such as for use in the prevention or treatment of acondition characterized by pathological pain, chronic pain,neuroinflammation, neurodegeneration, and/or local or systemicinflammation. In this regard, “chronic” can be regarded as persisting atleast 1, 2, 3, 4, 5, 6, 10, or 12 months, or even at least 1, 2, 3, 4,or 5 years. Said condition may be characterized by visceral ornon-visceral inflammatory pain, visceral or non-visceral nociceptivepain, peripheral or central neuropathic pain, mixednociceptive-neuropathic pain, neuro-inflammation, and/orneuro-degeneration, and/or may be selected from the group consisting ofpost-operative orthopedic surgery pain, musculoskeletal pain, irritablebowel syndrome, inflammatory bowel disease, rheumatoid arthritis,ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia,post-traumatic or post-operative peripheral neuropathy, diabeticperipheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, painful peripheral neuropathy, nerveentrapment syndrome, chemotherapy-associated pain, chemotherapy-inducedallodynia, chemotherapy-induced peripheral neuropathy, complex regionalpain syndrome, post-spinal injury pain, post-stroke pain, multiplesclerosis, low back pain, osteoarthritis, cancer pain, chronic visceralpain, fibromyalgia, polymyalgia rheumatica, chronic widespread pain,myofascial pain syndrome, Alzheimer's disease and Parkinson's disease,Huntington's disease, and/or amyotrophic lateral sclerosis, or multiplesclerosis.

In an aspect, the invention relates to a fusion protein as taught hereinfor use in the prevention or treatment of a clinical condition in amammal, such as a human, for which IL4 or IL13 is indicated.

The invention is also concerned with a fusion protein as taught hereinfor use in the prevention or treatment of a clinical condition in amammal, such as a human, for which IL10 or IL27 or IL33 or TGFβ1 orTGFβ2 is indicated.

Finally, the invention teaches a vector for use in the prevention ortreatment of a condition characterized by chronic pain,neuroinflammation, neurodegeneration, and/or local or systemicinflammation. Said condition may be characterized by visceral ornon-visceral nociceptive pain, peripheral or central neuropathic pain,or mixed nociceptive-neuropathic pain, neuro-inflammation, and/orneuro-degeneration, and/or may be selected from the group consisting ofpost-operative orthopedic surgery pain, musculoskeletal pain, irritablebowel syndrome, inflammatory bowel disease, rheumatoid arthritis,ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia,post-traumatic or post-operative peripheral neuropathy, diabeticperipheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, painful peripheral neuropathy, nerveentrapment syndrome, chemotherapy-associated pain, complex regional painsyndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis,low back pain, osteoarthritis, cancer pain, chronic visceral pain,fibromyalgia, polymyalgia rheumatica, myofascial pain syndromes,Alzheimer's disease and Parkinson's disease, Huntington's disease,and/or amyotrophic lateral sclerosis, or multiple sclerosis.

BRIEF DESCRIPTION OF THE FIGURES RELATED TO THE INVENTION

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present disclosure can be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the disclosure are utilized, and theaccompanying drawings of which:

FIG. 1. Regulatory (e.g., anti-inflammatory) cytokines are required forresolution of chemotherapy-induced pain. Mice were intraperitoneallyinjected with 2 mg/kg paclitaxel at day 0 and 2 to induce transientpainful chemotherapy-induced polyneuropathy. From day 6 on, micereceived daily intrathecal injections of neutralizing antibodies toendogenous IL4 (n=4; open downward triangles), or IL13 (n=4; closedupward triangles) for 5 days (5 μg antibody per injection). As acontrol, isotype IgG was injected intrathecally in mice treated withpaclitaxel (n=3; dotted line with open circles). As another control,mice were pretreated with vehicle instead of paclitaxel and control IgG(n=3; closed line with closed circles). Pain-like behavior was followedover time by measuring mechanical sensitivity to touch using von Freyhairs. Note that a lower 50% threshold indicates increased sensitivity.Data represent mean and standard error of the mean. Statisticaldifferences are indicated as *p<0.05, **p<0.01, ***p<0.001 betweenanti-IL13 IgG versus control IgG treated mice. x p<0.05, xx p<0.01between anti-IL4 IgG versus control IgG treated mice.

FIG. 2. IL13 attenuates paclitaxel-induced damage to neurons. Primarysensory neurons were cultured and treated overnight with Paclitaxel (1μM) to induce neuronal damage that was quantified by measuring theneurite length after β3-tubulin staining. Vehicle (−) or individualcytokines (50 ng/ml) were added during treatment with thechemotherapeutic drug, and the average length of neurons was measured.Data represent mean and standard error of the mean of the neurite lengthin microns of >10 cells measured in at least 2 experiments.

FIG. 3. Characterization of recombinant IL4/IL13 fusion protein.His-tagged IL4/IL13 fusion protein was expressed in HEK293 cells,purified using HIS-Select Nickel Affinity chromatography, and analyzedwith High Pressure Size Exclusion Chromatography (HP-SEC). The HP-SECprofile indicates a homogenous monomeric preparation. Insert showsSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)analysis of starting material (load), flow through (FT), washing buffer(wash) and eluate of the His-tag purification column. The gel wasstained with Coomassie Blue. Note that IL4/IL13 fusion protein migratesas a smear at 37 kDa.

FIG. 4. IL4/IL13 fusion protein relieves paclitaxel-induced persistentmechanical allodynia. Paclitaxel (8 mg/kg) was administeredintraperitoneally to C57BL/6 mice on days 0, 2, 4 and 6 (grey symbols onthe X-axis) to induce persistent chemotherapy-induced polyneuropathy.IL4/IL13 fusion protein (0.3 [open circle], 1 [open triangle] or 3μg/mouse [open square]; n=4/group) or vehicle (n=4) was administeredintrathecally at day 8, and the course of mechanical allodynia wasfollowed over time using von Frey hairs. Data is represented asmean±SEM. Statistics of the data were analysed with two-way ANOVAfollowed by Tukey's multiple comparisons test. *, **, ***=p<0.05,p<0.01, and 0.001, 0.3 μg IL4/IL13 fusion protein versus vehiclerespectively. &,=p<0.05, 3 μg IL4/IL13 fusion protein versus vehicle.x=p<0.05, 1 μg IL4/IL13 fusion protein versus vehicle.

FIG. 5. IL4/IL13 fusion protein has a longer lasting effect on painfulpaclitaxel-induced neuropathy in mice than IL4/IL10 fusion protein. Micereceived 4 intraperitoneal injections of 8 mg/kg paclitaxel every otherday (grey symbols on X-axis) to induce persisting painfulchemotherapy-induced polyneuropathy. At day 8, mice received a singleintrathecal injection of IL4/IL13 fusion protein (0.7 μg; open circles,n=4), IL4/IL10 fusion protein (0.7 μg; open triangles, n=3), thecombination of wildtype IL4 and IL13 (0.35 μg/cytokine; open squares,n=4) or vehicle (closed circles, n=4). Pain-like behavior was testedwith von Frey hairs (see FIG. 1). Note that a single administration ofIL4/IL13 fusion protein results in a sustained alleviation of pain(e.g., permanently resolves pain), whereas IL4/IL10 fusion protein has atemporary effect lasting 2 days in this experiment.

FIG. 6. IL4/IL13 fusion protein protects against paclitaxel-inducednerve damage in mice. Mice received 4 intraperitoneal injections of 8mg/kg paclitaxel every other day to induce persisting painfulchemotherapy-induced polyneuropathy. On day 8, they were treated with asingle intrathecal injection of IL4/IL13 fusion protein (0.7 μg), orvehicle. On day 15, the length of intraepidermal nerve fibers in the pawskin was determined upon immunofluorescent visualization with theneuronal marker PGP9.5. The data of mice not treated withchemotherapeutic drug (black bar; n=4), or injected with paclitaxel andsubsequently treated with vehicle (−; n=6) or IL4/IL3 fusion protein(IL4-13; n=4), are shown.

FIG. 7. IL4/IL13 fusion protein protects cultured neurons againstchemotherapy induced damage better than IL4/IL10 fusion protein or thecombination of IL4 and IL13. Primary sensory neurons were culturedovernight in presence of paclitaxel (1 μM) to induce neuronal damagethat was quantified by measuring the neurite length upon β3-tubulinstaining. Vehicle (−) or IL4/IL13 fusion protein (IL4-13), IL4/IL10fusion protein (IL4-10) or the combination of IL4 and IL13 (IL4+IL13)were added at equimolar concentrations during incubation with thechemotherapeutic drug. Neurons cultured in absence of paclitaxel andcytokines are shown for comparison (black bar).

FIG. 8. IL4/IL13 cures oxaliplatin-induced polyneuropathy in micewhereas IL4 or IL13 only have a partial transient effect. Oxaliplatin (3mg/kg) was daily injected intraperitoneally in mice for 5 days followedby 5 days no treatment and another 5 days of an oxaliplatin treatmentcycle (grey symbols on X-axis). On the day after the last oxaliplatininjection animals received an intrathecal injection of IL4/IL13 fusionprotein (0.3 μg; open circles, n=4) or the wild-type cytokines (0.15 μg;n=4, rectangles for IL4 and triangles for IL13); or vehicle only (closedcircles). Pain was measured with von Frey test.

FIG. 9. IL4/IL13 fusion protein, but not the combination of IL4 andIL13, protects cultured neurons against oxaliplatin-induced damage.Primary sensory neurons were cultured and treated overnight withoxaliplatin (5 μg/ml). Neuronal damage was then quantified by measuringthe neurite length upon β3-tubulin staining. Vehicle (−) or IL4/IL13fusion protein or the combination of IL4 and IL13 (IL4+IL13) were addedat equimolar concentrations during incubation with the chemotherapeuticdrug. Neurons cultured in absence of oxaliplatin and cytokines are shownfor comparison (black bar).

FIG. 10. Cytokine-receptor subunits of IL10, IL4, IL13, IL33, IL27,TGFβ1, and TGFβ2 are expressed in the dorsal root ganglia of human andmouse. To evaluate whether cytokine receptors targeted by fusionproteins of the present invention are expressed by the sensory system,RNAseq data of cytokine receptor subunits for IL10, IL4, IL13, IL33,IL27 TGFβ1, and TGFβ2 in the dorsal root ganglia and spinal cord wereextracted from the data base by Ray et al. (Pain 2018; 159:1325-1345) asavailable onwww_utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?go. RNAsequencing data are expressed as transcripts per million. Forcomparison, data for expression of the receptors in whole blood are alsogiven.

FIG. 11. Characterization of fusion proteins. Purified fusion proteinswere analysed on a 4-12% gradient NuPage™ polyacrylamide gel undernon-reducing and reducing conditions, and bands were visualized byCoomassie protein stain.

FIG. 12. Two IL4/IL13 fusion proteins of the disclosure protect neuronsagainst chemotherapy induced neuron damage. Primary sensory neurons werecultured for 24 h in the presence of paclitaxel (1 μM), and differentconcentrations of each fusion protein or equimolar doses of IL13 or thecombination of unlinked cytokines. The inhibition of paclitaxel-induceddecrease in neurite length was calculated. The fusion protein labeledIL4/IL13 comprises SEQ ID NO: 4. The fusion protein labeledIL4/IL13_(SKP) comprises SEQ ID NO: 16. Data are shown as mean±SEM. Dataare analysed with a two-way ANOVA mixed-effects analysis followed byTukey's multiple comparison test.

FIG. 13. IL10/IL13 protect neurons against chemotherapy induced neurondamage. Primary sensory neurons were cultured for 24 h in the presenceof paclitaxel (1 μM), and different concentrations of each fusionprotein or equimolar doses of IL13 or the combination of unlinkedcytokines. The inhibition of paclitaxel-induced decrease in neuritelength was calculated. Data are shown as mean±SEM. Data are analysedwith a two-way ANOVA mixed-effects analysis followed by Tukey's multiplecomparison test.

FIG. 14. Protective effects of IL13/IL13 and IL27/IL13 againstchemotherapy induced neuron damage. Primary sensory neurons werecultured for 24 h in the presence of paclitaxel (1 μM), and differentconcentrations of each fusion protein or an equimolar dose of IL13. Theinhibition of paclitaxel-induced decrease in neurite length wascalculated. Data are shown as mean±SEM. Data are analysed with a two-wayANOVA mixed-effects analysis followed by Tukey's multiple comparisontest.

FIG. 15. An IL13-containing fusion protein of the disclosure elicits adistinct kinase activity profile in dorsal root ganglia (DRG) cellscompared to a combination of unlinked cytokines. PamGene kinase activityprofiling was performed to assess global protein tyrosine kinases (PTK)activity in homogenates of lumbar DRGs isolated from mice withpersistent paclitaxel-induced peripheral neuropathy after IL4/IL13fusion protein, IL4+IL13 (combination of unlinked cytokines), andvehicle administration. Kinomic profiles were assessed at 60 minutesafter intrathecal administration of the IL4/IL13 fusion protein, thecombination of cytokines, or vehicle (PBS). Peptides are shown that weredifferentially phosphorylated based on one-way ANOVA analysis betweenIL4/IL13, IL4+IL13, and vehicle-treated mice compared to naive mice(untreated; no paclitaxel, no intrathecal injection). Black indicates nosignificant changes, while color indicates decreased phosphorylation.

FIG. 16. Altered kinase activity in dorsal root ganglia (DRG) cells offemale mice treated with IL4/IL13 compared to a combination of unlinkedcytokines. PamGene kinase activity profiling was performed to assessglobal protein tyrosine kinases (PTK) activity in homogenates of lumbarDRGs isolated from female mice with persistent paclitaxel-induced CIPNafter IL4/IL13 fusion protein or IL4+IL13 (combination of unlinkedcytokines) administration. The graph shows the predicted upstreamkinases inferred from the differentially phosphorylated peptidesubstrates identified by unpaired t-test comparison between samples fromIL4/IL13 fusion protein-treated females and IL4+IL13-treated females(n=3 animals per group). The graph is sorted with the highestspecificity scores at the top, and the lowest specificity scores at thebottom.

FIG. 17. Altered kinase activity in dorsal root ganglia (DRG) cells ofmale mice treated with IL4/IL13 compared to a combination of unlinkedcytokines. PamGene kinase activity profiling was performed to assessglobal protein tyrosine kinases (PTK) activity in homogenates of lumbarDRGs isolated from male mice with persistent paclitaxel-induced CIPNafter IL4/IL13 fusion protein or IL4+IL13 (combination of unlinkedcytokines) administration. The graph shows the predicted upstreamkinases inferred from the differentially phosphorylated peptidesubstrates identified by unpaired t test comparison between samples fromIL4/IL13 fusion protein-treated males and IL4+IL13-treated males (n=3animals per group). The graph is sorted with the highest specificityscores at the top, and the lowest specificity scores at the bottom.

FIGS. 18A-18D. Size exclusion chromatography of IL4/IL13, IL10/IL13,IL27/IL13, and IL13/IL13 fusion proteins. Size exclusion chromatographywas performed for IL4/IL13, IL10/IL13, IL27/IL13, and IL13/IL13 fusionproteins of the disclosure. FIG. 18A: Size exclusion chromatography ofan IL4/IL13 fusion protein containing SEQ ID NO: 16. FIG. 18B: Sizeexclusion chromatography of an IL13/IL13 fusion protein containing SEQID NO: 20. FIG. 18C: Size exclusion chromatography of an IL27/IL13fusion protein containing SEQ ID NO: 19. FIG. 18D: Size exclusionchromatography of an IL10/IL13 fusion protein containing SEQ ID NO: 17.

DETAILED DESCRIPTION OF THE INVENTION

General Definitions

The term “nucleic acid molecule” (or “nucleic acid sequence”,“polynucleotide”, or “nucleotide sequence”) refers to a DNA or RNAmolecule in single or double stranded form, particularly a DNA encodinga protein according to the invention. An “isolated nucleic acidsequence” refers to a nucleic acid sequence which is no longer in thenatural environment from which it was isolated, e.g., the nucleic acidsequence in a bacterial host cell or in the plant nuclear or plastidgenome.

The terms “protein” or “polypeptide” are used interchangeably and referto molecules consisting of one or several chains of amino acids, withoutreference to a specific mode of action, size, three-dimensionalstructure or origin. An “isolated protein” is used to refer to a proteinwhich is no longer in its natural environment, for example in vitro orin a recombinant mammalian, bacterial, or plant host cell.

The term “fusion protein” refers to a protein or polypeptide that has anamino acid sequence from or derived from two or more proteins. Thefusion protein may also include linking regions or a linker of aminoacids between amino acid portions from or derived from separateproteins. The fusion protein also refers to a molecule that has an aminoacid sequence from or derived from two or more proteins which arenon-covalently bound, or connected via chemical crosslinkers (e.g.,covalently linked), with or without a spacer. A fusion protein can be apolypeptide construct.

The terms IL4, IL10, IL13, IL27, IL33, TGFβ1, and TGFβ2 preferably referto the wild-type sequences of these respective cytokines, and/or tomutated variants thereof capable of binding to at least one of theirrespective cytokine receptors or receptor subunits (e.g., preferably atleast two).

The terms “TGFβ”, “TGFβ1/2” and “TGFβ(1 or 2)” are used to refer toTGFβ1 and/or TGFβ2.

The term “IL4/IL13 fusion protein” refers to a fusion polypeptidecomprising at least IL4 and IL13, optionally coupled to one another viaa linker. The fusion protein may comprise additional polypeptidesequences, e.g., a signal sequence, a His-tag, targeting sequence(s), anantibody Fc fragment, an extracellular matrix-binding polypeptide, orany combination thereof.

The term “IL10/IL13 fusion protein” refers to a fusion polypeptidecomprising at least IL10 and IL13, optionally coupled to one another viaa linker. The fusion protein may comprise additional polypeptidesequences, e.g., a signal sequence, a His-tag, targeting sequence(s) oran antibody Fc fragment, an extracellular matrix-binding polypeptide, orany combination thereof.

The term “IL33/IL13 fusion protein” refers to a fusion polypeptidecomprising at least IL33 and IL13, optionally coupled to one another viaa linker. The fusion protein may comprise additional polypeptidesequences, e.g., a signal sequence, a His-tag, targeting sequence(s) oran antibody Fc fragment, an extracellular matrix-binding polypeptide, orany combination thereof.

The term “TGFβ1/IL13 fusion protein” refers to a fusion polypeptidecomprising at least TGFβ1 and IL13, optionally coupled to one anothervia a linker. The fusion protein may comprise additional polypeptidesequences, e.g., a signal sequence, a His-tag, targeting sequence(s) oran antibody Fc fragment, an extracellular matrix-binding polypeptide, orany combination thereof.

The term “TGFβ2/IL13 fusion protein” refers to a fusion polypeptidecomprising at least TGFβ2 and IL13, optionally coupled to one anothervia a linker. The fusion protein may comprise additional polypeptidesequences, e.g., a signal sequence, a His-tag, targeting sequence(s) oran antibody Fc fragment, an extracellular matrix-binding polypeptide, orany combination thereof.

The term “IL13/IL13 fusion protein” refers to a fusion polypeptidecomprising at least two IL13 molecules, optionally coupled to oneanother via a linker. The fusion protein may comprise additionalpolypeptide sequences, e.g., a signal sequence, a His-tag, targetingsequence(s) or an antibody Fc fragment, an extracellular matrix-bindingpolypeptide, or any combination thereof.

As used herein, a “linker” means a polypeptide used to couple twoproteins or polypeptides, in casu IL4 (or IL10 or IL27 or IL33 or TGFβor IL13) and IL13. The linker typically is a stretch of amino acids,e.g., predominantly glycine and/or serine. In an embodiment, the linkeris a stretch of amino acids having a length of up to 100 amino acids,such as from about 2, 5, 7, 10, 15 amino acids up to about 15, 20, 25,30, 35, 50, 75, or 100 amino acids, preferably comprising predominantlyserine and glycine residues.

As used herein, “interleukin-13” (IL13) preferably refers to anymammalian IL13, such as human IL13, mouse IL13, or an active species orvariant (e.g., allelic variant), (functional) fragment or derivativethereof.

As used herein, “interleukin-4” (IL4) preferably refers to any mammalianIL4, such as human IL4, mouse IL4, or an active species or variant(e.g., allelic variant), (functional) fragment or derivative thereof.

As used herein, “interleukin-10” (IL10) preferably refers to anymammalian IL10, such as human IL10, mouse IL10, or an active species orvariant (e.g., allelic variant), (functional) fragment or derivativethereof.

As used herein, “interleukin-27” (IL27) can refer to any mammalian IL27,such as human IL27, mouse IL27, or an active species or variant (e.g.,allelic variant), (functional) fragment or derivative thereof. In somecases, IL27 refers to IL27 subunit alpha (IL27A), for example, humanIL27A. In some cases, IL27 refers to IL27 subunit beta (IL27B), forexample, human IL27B. In some cases, IL27 refers to IL27A and IL27B.

As used herein, “interleukin-33” (IL33) preferably refers to anymammalian IL33, such as human IL33, mouse IL33, or an active species orvariant (e.g., allelic variant), (functional) fragment or derivativethereof.

As used herein, “transforming growth factor β1” (TGFβ1) preferablyrefers to any mammalian TGFβ1, such as human TGFβ1, mouse TGFβ1, or anactive species or variant (e.g., allelic variant), (functional) fragmentor derivative thereof.

As used herein, “transforming growth factor β2” (TGFβ2) preferablyrefers to any mammalian TGFβ2, such as human TGFβ2, mouse TGFβ2, or anactive species or variant (e.g., allelic variant), (functional) fragmentor derivative thereof.

When describing a cytokine, the term “wild type” refers to a cytokinewith an amino acid sequence that is naturally occurring and encoded by agermline genome of a given species. A species can have one wild typesequence, or two or more wild type sequences (for example, with onecanonical wild type sequence and one or more non-canonical wild typesequences). A wild type cytokine sequence can include a sequence that istruncated at the N and/or C terminus relative to the sequence encoded byan open reading frame. A wild type cytokine sequence can be a matureform of a cytokine that has been processed to remove N-terminal and/orC-terminal residues. A wild type cytokine can lack a signal peptide orcan include a signal peptide (e.g., a signal peptide can be added to theN-terminus of the wild type cytokine). When describing a cytokine, theterm “derivative” refers to a cytokine with an amino acid sequence thatdiffers from a wild type sequence by one or more amino acids, forexample, containing one or more amino acid insertions, deletions, orsubstitutions relative to a wild type sequence. A cytokine derivativebinds to at least one subunit of the corresponding native receptor forthe wild type cytokine and elicits signaling and/or cytokine activity.The binding affinity, signaling, and/or cytokine activity of a cytokinederivative can be the same or different than the corresponding wild typecytokine.

“Functional”, in relation to the fusion proteins of the presentinvention (or variants or fragments thereof), refers to the capabilityto display both IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2 or otherregulatory cytokine, e.g., anti-inflammatory cytokine) and IL13functionality, for example, the ability to bind to at least one receptorsubunit that a wild type version of the cytokine binds.

Assays to assess the functional activity of these cytokines are wellknown to those skilled in the art. For example, a functional assay forIL4 and IL10 is the lipopolysaccharide (LPS) induced TNF release inwhole blood in presence of anti-IL10 antibody²⁶. A functional assay forIL13 is the proliferation of TF1 human erythroleukemic cells²⁷. An assayfor IL33 function is IL6 production by the mast cell line MC/9. An assayfor TGFβ1 or TGFβ2 is inhibition of IL4-dependent growth of mouse T-cellline HT-2. A functional assay for IL27 can comprise IL6 production byLPS-stimulated THP-1 macrophages.

The term “gene” means a DNA sequence comprising a region (transcribedregion), which is transcribed into an RNA molecule (e.g. a mRNA) in acell, operably linked to suitable regulatory regions (e.g. a promoter).A gene may thus comprise several operably linked sequences, such as apromoter, a 5′ leader sequence comprising e.g. sequences involved intranslation initiation, a (protein) coding region (cDNA or genomic DNA),introns, and a 3′ non-translated sequence comprising e.g. transcriptiontermination sites.

“Expression of a gene” refers to the process wherein a DNA region, whichis operably linked to appropriate regulatory regions, particularly apromoter, is transcribed into an RNA, which is biologically active, i.e.which is capable of being translated into a biologically active proteinor peptide (or active peptide fragment). “Expression of a polypeptide”additionally refers to a process wherein an mRNA is translated into aprotein product, which may or may not be secreted.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active in most tissuesunder most physiological and developmental conditions. An “inducible”promoter is a promoter that is physiologically (e.g. by externalapplication of certain compounds) or developmentally regulated. A“tissue specific” promoter is only active in specific types of tissuesor cells.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter, or rather atranscription regulatory sequence, is operably linked to a codingsequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous.

A “nucleic acid construct” or “vector” is herein understood to mean aman-made nucleic acid molecule resulting from the use of recombinant DNAtechnology and which is used to deliver exogenous DNA into a host cell.Vectors usually comprise further genetic elements to facilitate theiruse in molecular cloning, such as e.g. selectable markers, or multiplecloning sites (see below).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they (whenoptimally aligned by for example the programs GAP or BESTFIT usingdefault parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength, maximizing the number of matches and minimizes the number ofgaps. Generally, the GAP default parameters are used, with a gapcreation penalty=50 (nucleotides)/8 (proteins) and gap extensionpenalty=3 (nucleotides)/2 (proteins). For nucleotides the defaultscoring matrix used is nwsgapdna and for proteins the default scoringmatrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignments and scores for percentage sequence identity may bedetermined using computer programs, such as the GCG Wisconsin Package,Version 10.3, available from Accelrys Inc., 9685 Scranton Road, SanDiego, Calif. 92121-3752 USA, or EmbossWin version 2.10.0 (using theprogram “needle”). Alternatively, percent similarity or identity may bedetermined by searching against databases, using algorithms such asFASTA, BLAST, etc. Preferably, the sequence identity refers to thesequence identity over the entire length of the sequence.

A “host cell” or a “recombinant host cell” or “transformed cell” areterms referring to a new individual cell (or organism) arising as aresult of at least one nucleic acid molecule, especially comprising anucleic acid molecule encoding a desired protein. The host cell ispreferably a mammalian cell, plant cell or a bacterial cell. The hostcell may contain the nucleic acid molecule or vector of the presentinvention as an extra-chromosomally (episomal) replicating molecule, ormore preferably, comprises the nucleic acid molecule or vector of thepresent invention integrated in the genome of the host cell.

The term “selectable marker” is a term familiar to one of ordinary skillin the art and is used herein to describe any genetic entity which, whenexpressed, can be used to select for a cell or cells containing theselectable marker. Selectable marker gene products confer for exampleantibiotic resistance or nutritional requirements.

The term “nervous system cell” refers to a cell that is found within thecentral nervous system or peripheral nervous system. A nervous systemcell can be a neuron, a central nervous system cell, a peripheralnervous system cell, a glial cell, a microglial cell, an astrocyte, aschwann cell, a satellite glial cell, an oligodendrocyte, aninfiltrating cell, an infiltrating immune cell, an infiltrating myeloidcell, an infiltrating lymphoid cell, an infiltrating macrophage, aninfiltrating neutrophil, an infiltrating lymphocyte, an infiltrating Tcell, an infiltrating B cell, or an infiltrating natural killer cell. Aneuron can be, for example, a sensory neuron, a somatosensory neuron, avisceral sensory neuron, a nociceptor, and/or an autonomic neuron.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. It also encompasses the more limiting verb “to consistof”. In addition, reference to an element by the indefinite article “a”or “an” does not exclude the possibility that more than one of theelements is present, unless the context clearly requires that there beone and only one of the elements. The indefinite article “a” or “an”thus usually means “at least one”. It is further understood that, whenreferring to “sequences” herein, generally the actual physical moleculeswith a certain sequence of subunits (e.g. amino acids) are referred to.

Proteins, Nucleic Acid Sequences, Vectors and Host Cells of theInvention

The present inventors provide a fusion protein comprising an IL13protein and a regulatory cytokine, for example, a protein chosen fromIL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or IL13 itself, optionallyphysically fused together via a linker. Regulatory cytokines of thedisclosure include, but are not limited to, IL4, IL10, IL13, IL27, IL33,TGFβ1, and TGFβ2. Particularly, the fusion protein of the presentinvention was found to have a superior activity in a treatment for acondition disclosed herein (e.g., neuropathic pain) over its individualcounterparts, i.e., regulatory cytokine (e.g., IL4 or IL10 or IL27 orIL33 or TGFβ1 or TGFβ2) and IL13 separately. Specifically, it was foundthat, upon intrathecal administration, the fusion protein of the presentinvention has a long-lasting analgesic effect on neuropathic pain, andabrogates allodynia associated with chemotherapy-induced neuropathy. Theinventors unexpectedly observed that this analgesic effect of the fusionprotein of the present invention lasted longer than that of a previouslydescribed fusion protein of IL4 and IL10^(19,23,26). Surprisingly, thefusion protein of the present invention also improvedchemotherapy-associated neuro-degeneration. It prevented the shorteningof neurites in vitro upon incubation with chemotherapeutic drugs, andwas more potent regarding this effect than a fusion protein of IL4 andIL10, or the combination of IL4 (or IL10 or IL27 or IL33 or TGFβ1 orTGFβ2) and IL13. The latter surprising finding indicates a unique effectof the fusion protein over its individual cytokines or the combinationof these. In vivo, the fusion protein of the present inventionattenuated the decrease of intraepidermal nerve fibers uponadministration of chemotherapeutic drugs.

In one embodiment of the invention, nucleic acid sequences and aminoacid sequences of IL4/IL13 fusion proteins or IL10/IL13 fusion proteinsor IL27/IL13 fusion proteins or IL33/IL13 fusion proteins or TGFβ1/IL13fusion proteins or TGFβ2/IL13 or IL13/IL13 fusion proteins are provided(including variants, derivatives, and fragments thereof). The IL4/IL13fusion proteins or IL10/IL13 fusion proteins or IL27/IL13 fusionproteins or IL33/IL13 fusion proteins, as well as derivatives,functional fragments and variants thereof, display IL4 (or IL10 or IL27or IL33 or TGFβ1 or TGFβ2) activity as well as IL13 activity.

In some cases, fusion proteins disclosed herein contain two cytokines,C1 and C2, and are referred to in the format C1/C2 or C1-C2. The orderin which the cytokines are presented is not limiting and does notnecessarily infer the orientation of the cytokines. For example, C1/C2or C1-C2 can contain cytokine C1 on the C-terminal side of C2 or on theN-terminal side of C2. Similarly, a cytokine referred to as C1/C2 can bethe same as a cytokine referred to as C2/C1 unless otherwise specified.

In one aspect, a fusion protein comprising IL4 (or IL10 or IL13 or IL27or IL33 or TGFβ1 or TGFβ2) and IL13 is provided.

Interleukin 13

A fusion protein disclosed herein can comprise an IL13 protein, or avariant, derivative, or fragment thereof operably linked or directly orindirectly fused to a regulatory cytokine or a variant or derivativethereof. The IL13 protein is preferably a mammalian IL13 protein, suchas a human IL13, or mouse IL13. Non-limiting examples of amino acidsequences representing human IL13 are set forth in SEQ ID NO:2 and SEQID NOs: 9-15. Variants of IL13 include, for example, proteins having atleast 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more,such as 100%, amino acid sequence identity, to SEQ ID NO:2 or any one ofSEQ ID NOs: 9-15, preferably over the entire length. Amino acid sequenceidentity is preferably determined by pairwise alignment using theNeedleman and Wunsch algorithm and GAP default parameters as definedabove. Variants, derivatives, and fragments thereof also includeproteins having IL13 activity, which have been derived, by way of one ormore amino acid substitutions, deletions or insertions, from thepolypeptide having the amino acid sequence of SEQ ID NO:2 or any one ofSEQ ID NOs: 9-15. Preferably, such proteins comprise from 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35,30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL13 of the disclosure (e.g., an IL13 variant,derivative, or fragment thereof) can comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least or at least 50 amino acid substitutions, deletions, orinsertions relative to an IL13 sequence disclosed herein (e.g., a wildtype IL13 sequence).

In some embodiments, an IL13 of the disclosure (e.g., an IL13 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to an IL13sequence disclosed herein (e.g., a wild type IL13 sequence).

In some embodiments, an IL13 sequence of the disclosure (e.g., an IL13variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to an IL13 sequence disclosed herein(e.g., a wild type IL13 sequence).

In some embodiments, an IL13 sequence of the disclosure (e.g., an IL13variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to an IL13 sequencedisclosed herein (e.g., a wild type IL13 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

An IL13 of the disclosure can comprise a wild type IL13 sequence.Non-limiting examples of wild type IL13 sequences include SEQ ID NOs: 2and 9-15. SEQ ID NO: 12 can be a canonical wild type IL13 sequence ofthe disclosure.

An IL13 of the disclosure can comprise an IL13 variant, derivative, orfragment thereof with one or more amino acid substitutions. For example,an IL13 variant, derivative, or fragment thereof can comprise an aminoacid substitution at position L10, E12, R11, I14, E15, E16, V18, R65,S68, R86, D87, T88, K89, D98, L101, L103, K104, K105 L106, F107, R108,R111, F114, N113, or a combination thereof of SEQ ID NO: 2 or SEQ ID NO:12. In some embodiments, an IL13 variant, derivative, or fragmentthereof comprises a substitution that is L10F; L10I; L10V; L10A; L10D;L10T; L10H; R11S; R11N; R11H; R11L; R11I; I14L; I14F; I14V; I14M; V18L;V18F; V18I; E12A; R65D; R86K; R86T; R86M; D87E; D87K; D87R; D87G; D87S;T88S, T88I; T88K; T88R; K89R; K89T; K89M; L101F; L101I; L101Y; L101H;L101N; K104R; K104T; K104M; K105T; K105A; K105R; K105E; F107L; F1071;F107V; F107M; R108K; R108T; R108M; E12K, E12I, E12C, E12S, E12R, E12Y,E12D, E15K, E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A,F107Y, R108D, R111D, F114D, N113D, or a combination thereof relative toSEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions L10H, R86T,D87G, T88R, and R108K relative to SEQ ID NO: 2 or SEQ ID NO: 12. In someembodiments, an IL13 variant, derivative, or fragment thereof comprisesthe substitutions L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108Krelative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13variant, derivative, or fragment thereof comprises the substitutionsR11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T relative toSEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions L10V, K89R,L101 N, K105E, and R108T relative to SEQ ID NO: 2 or SEQ ID NO: 12. Insome embodiments, an IL13 variant, derivative, or fragment thereofcomprises the substitutions L10D, R11I, V18I, R86K, D87K, K89R, andR108K relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, anIL13 variant, derivative, or fragment thereof comprises thesubstitutions L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, andR108K relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, anIL13 variant, derivative, or fragment thereof comprises thesubstitutions L10V, K89R, L101 N, K105E, and R108T relative to SEQ IDNO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions R11S, I14M,T88S, L101 N, K105A, and R108K relative to SEQ ID NO: 2 or SEQ ID NO:12. In some embodiments, an IL13 variant, derivative, or fragmentthereof comprises the substitutions L10H, R11 L, V18I, R86K, D87E, K89R,L101N, K105T, and R108K relative to SEQ ID NO: 2 or SEQ ID NO: 12. Insome embodiments, an IL13 variant, derivative, or fragment thereofcomprises the substitutions L10H, R86T, D87G, T88R, and R108K relativeto SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions L10A, V18F,R86K, D87K, K89R, L101I, K104R, and R108K relative to SEQ ID NO: 2 orSEQ ID NO: 12. In some embodiments, an IL13 variant, derivative, orfragment thereof comprises the substitutions L10T or L10D; R11I; V18I;R86K; D87K or D87G; T88S; K89R; L101Y; K104R; K105T; and R108K relativeto SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions L10A orL10V; R86T; D87G; T88K; K89R; L101N; K104R; K105A or K105E; and R108K orR108T relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, anIL13 variant, derivative, or fragment thereof comprises thesubstitutions L10V, V18I, D87S, D88S, L101F, K104R, and K105T relativeto SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions R11S, V18I,R86K, D87G, T88S, K89M, L101Y, K104R, and K105T relative to SEQ ID NO: 2or SEQ ID NO: 12. In some embodiments, an IL13 variant, derivative, orfragment thereof comprises the substitutions L10V, V18I, D87S, T88S,L101F, K104R, and K105T relative to SEQ ID NO: 2 or SEQ ID NO: 12. Insome embodiments, an IL13 variant, derivative, or fragment thereofcomprises the substitutions L10V or L10I; D87S; T88S; K89R; L101H orL101F; K104R; and K105T relative to SEQ ID NO: 2 or SEQ ID NO: 12. Insome embodiments, an IL13 variant, derivative, or fragment thereofcomprises the substitutions L10I; V18I; R86T; D87G; T88S; K89R; L101Y,L101H; K104R; and K105A relative to SEQ ID NO: 2 or SEQ ID NO: 12. Insome embodiments, an IL13 variant, derivative, or fragment thereofcomprises the substitutions L10V; V18I; D87S; T88S; L101F; K104R; andK105T relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, anIL13 variant, derivative, or fragment thereof comprises thesubstitutions V18I, R86T, D87G, T88S, L101Y, K104R, and K105A relativeto SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions R11I, V18I,R86K, D87G, T88S, L101H, K104R, K105A, and F107M relative to SEQ ID NO:2 or SEQ ID NO: 12. In some embodiments, an IL13 variant, derivative, orfragment thereof comprises the substitutions E12K and S68D relative toSEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions E12K andR108D relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, anIL13 variant, derivative, or fragment thereof comprises thesubstitutions E12K and R111D relative to SEQ ID NO: 2 or SEQ ID NO: 12.In some embodiments, an IL13 variant, derivative, or fragment thereofcomprises the substitutions E12Y and R65D relative to SEQ ID NO: 2 orSEQ ID NO: 12. In some embodiments, an IL13 variant, derivative, orfragment thereof comprises the substitutions E12Y and S68D relative toSEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, an IL13 variant,derivative, or fragment thereof comprises the substitutions E12K, R65Dand S68D relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments,an IL13 variant, derivative, or fragment thereof comprises thesubstitutions E12Y, R65D and S68D relative to SEQ ID NO: 2 or SEQ ID NO:12. In some embodiments, an IL13 variant, derivative, or fragmentthereof comprises the substitutions E12K, R65D, S68D and R111D relativeto SEQ ID NO: 2 or SEQ ID NO: 12.

In some embodiments, an IL13 variant, derivative, or fragment thereofdoes not contain a substitution at position L10, E12, R11, I14, E15,E16, V18, R65, S68, R86, D87, T88, K89, D98, L101, L103, K104, K105L106, F107, R108, R111, F114, or N113 relative to SEQ ID NO: 2 or SEQ IDNO: 12. In some embodiments, an IL13 variant, derivative, or fragmentthereof does not contain a L10F; L10I; L10V; L10A; L10D; L10T; L10H;R11S; R11N; R11H; R11L; R11I; I14L; I14F; I14V; I14M; V18L; V18F; V18I;E12A; R65D; R86K; R86T; R86M; D87E; D87K; D87R; D87G; D87S; T88S, T88I;T88K; T88R; K89R; K89T; K89M; L101F; L101I; L101Y; L101H; L101N; K104R;K104T; K104M; K105T; K105A; K105R; K105E; F107L; F107I; F107V; F107M;R108K; R108T; R108M; E12K, E12I, E12C, E12S, E12R, E12Y, E12D, E15K,E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A, F107Y, R108D,R111D, F114D, or N113D substitution.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure binds to an IL13 receptor subunit with about acomparable affinity as a wild type IL13 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold increasedaffinity compared to a wild type IL13 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold decreasedaffinity compared to a wild type IL13 sequence.

For example, an IL13 or IL13 variant, derivative, or fragment thereof ofthe disclosure can bind to an interleukin 13 receptor alpha 1(IL-13Rα1), interleukin 13 receptor alpha 2 (IL-13Rα2), interleukin 4receptor alpha (IL-4Rα), or a combination thereof with about acomparable affinity as a wild type IL13. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα1 with about a comparable affinity as a wild type IL13. Insome embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can bind to IL-13Rα2 with about a comparableaffinity as a wild type IL13. In some embodiments, an IL13 or IL13variant, derivative, or fragment thereof of the disclosure can bind toIL-4Rα with about a comparable affinity as a wild type IL13 sequence. Insome embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can bind to IL-13Rα1 and IL-13Rα2 with about acomparable affinity as a wild type IL13. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα1 and IL-4Rα with about a comparable affinity as a wild typeIL13. In some embodiments, an IL13 or IL13 variant, derivative, orfragment thereof of the disclosure can bind to IL-13Rα2 and IL-4Rα withabout a comparable affinity as a wild type IL13. In some embodiments, anIL13 or IL13 variant, derivative, or fragment thereof of the disclosurecan bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with about a comparableaffinity as a wild type IL13.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can bind to an IL13 receptor subunit with atleast a comparable affinity as a wild type IL13. For example, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto an interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13receptor alpha 2 (IL-13Rα2), interleukin 4 receptor alpha (IL-4Rα), or acombination thereof with at least a comparable affinity as a wild typeIL13. In some embodiments, an IL13 or IL13 variant, derivative, orfragment thereof of the disclosure can bind to IL-13Rα1 with at least acomparable affinity as a wild type IL13. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα2 with at least a comparable affinity as a wild type IL13. Insome embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can bind to IL-4Rα with at least a comparableaffinity as a wild type IL13 sequence. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα1 and IL-13Rα2 with at least a comparable affinity as a wildtype IL13. In some embodiments, an IL13 or IL13 variant, derivative, orfragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα withat least a comparable affinity as a wild type IL13. In some embodiments,an IL13 or IL13 variant, derivative, or fragment thereof of thedisclosure can bind to IL-13Rα2 and IL-4Rα with at least a comparableaffinity as a wild type IL13. In some embodiments, an IL13 or IL13variant, derivative, or fragment thereof of the disclosure can bind toIL-13Rα1, IL-13Rα2, and IL-4Rα with at least a comparable affinity as awild type IL13.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can bind to an IL13 receptor subunit with atmost a comparable affinity as a wild type IL13. For example, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto an interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13receptor alpha 2 (IL-13Rα2), interleukin 4 receptor alpha (IL-4Rα), or acombination thereof with at most a comparable affinity as a wild typeIL13. In some embodiments, an IL13 or IL13 variant, derivative, orfragment thereof of the disclosure can bind to IL-13Rα1 with at most acomparable affinity as a wild type IL13. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα2 with at most a comparable affinity as a wild type IL13. Insome embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can bind to IL-4Rα with at most a comparableaffinity as a wild type IL13 sequence. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα1 and IL-13Rα2 with at most a comparable affinity as a wildtype IL13. In some embodiments, an IL13 or IL13 variant, derivative, orfragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα withat most a comparable affinity as a wild type IL13. In some embodiments,an IL13 or IL13 variant, derivative, or fragment thereof of thedisclosure can bind to IL-13Rα2 and IL-4Rα with at most a comparableaffinity as a wild type IL13. In some embodiments, an IL13 or IL13variant, derivative, or fragment thereof of the disclosure can bind toIL-13Rα1, IL-13Rα2, and IL-4Rα with at most a comparable affinity as awild type IL13.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof can bind to an IL-13Rα1 with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL13 sequence. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof can bind to an IL-13Rα1with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL13 sequence.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof can bind to an IL-13Rα2 with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL13 sequence. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof can bind to an IL-13Rα2with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL13 sequence.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof can bind to an IL-4Rα with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL13 sequence. In some embodiments, an IL13 orIL13 variant, derivative, or fragment thereof can bind to an IL-4Rα withat least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL13 sequence.

In some embodiments, an IL13 or IL13 variant, derivative, or fragmentthereof of the disclosure can activate a native IL13 receptor. A nativeIL13 receptor can be, for example, a receptor comprising an IL-13Rα1subunit and an IL-4Rα subunit.

Interleukin 4

A fusion protein disclosed herein may comprise an IL4 protein, or avariant, derivative, or fragment thereof operably linked or directly orindirectly fused to an interleukin 13 or a variant or derivativethereof. The IL4 protein is preferably a mammalian IL4 protein, such asa human IL4, or mouse IL4. Non-limiting examples of amino acid sequencesof IL4 are set forth in SEQ ID NO:1 and SEQ ID NOs: 26-28. Variants ofIL4 include, for example, proteins having at least 70%, 75%, 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acidsequence identity to SEQ ID NO:1 or any one of SEQ ID NOs: 26-28,preferably over the entire length. Amino acid sequence identity ispreferably determined by pairwise alignment using the Needleman andWunsch algorithm and GAP default parameters as defined above. Variantsalso include proteins having IL4 activity, which have been derived, byway of one or more amino acid substitutions, deletions or insertions,from the polypeptide having the amino acid sequence of SEQ ID NO:1 orany one of SEQ ID NOs: 26-28. Preferably, such proteins comprise from 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50,45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions orinsertions. In some embodiments, an IL4 of the disclosure (e.g., an IL4variant, derivative, or fragment thereof) can comprise at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least or at least 50 amino acid substitutions,deletions, or insertions relative to an IL4 sequence disclosed herein(e.g., a wild type IL4 sequence).

In some embodiments, an IL4 of the disclosure (e.g., an IL4 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to an IL4 sequencedisclosed herein (e.g., a wild type IL4 sequence).

In some embodiments, an IL4 sequence of the disclosure (e.g., an IL4variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to an IL4 sequence disclosed herein(e.g., a wild type IL4 sequence).

In some embodiments, an IL4 sequence of the disclosure (e.g., an IL4variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to an IL4 sequencedisclosed herein (e.g., a wild type IL4 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

An IL4 of the disclosure can comprise a wild type IL4 sequence.Non-limiting examples of wild type IL4 sequences include SEQ ID NOs: 1and 26-28. A canonical wild type IL4 sequence of the disclosure can beSEQ ID NO: 1.

An IL4 of the disclosure can comprise an IL4 variant, derivative, orfragment thereof with one or more amino acid substitutions. For example,an IL4 variant, derivative, or fragment thereof can comprise an aminoacid substitution at position K117, T118, R121, E122, Y124, S125, S128,S129, or a combination thereof of SEQ ID NO: 1. In some embodiments, anIL4 variant, derivative, or fragment thereof comprises a substitutionthat is K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F,Y124D, S125F, S128G, S125R, S129A, or a combination thereof relative toSEQ ID NO: 1. In some embodiments, an IL4 variant, derivative, orfragment thereof comprises the substitutions K117R, T118V, R121Q, E122S,Y124W, S125F, S128G, and S129A relative to SEQ ID NO: 1. In someembodiments, an IL4 variant, derivative, or fragment thereof comprisesthe substitutions R121D and Y124D relative to SEQ ID NO: 1.

In some embodiments, an IL4 variant, derivative, or fragment thereofdoes not contain a substitution at position K117, T118, R121, E122,Y124, S125, S128, or S129, relative to SEQ ID NO: 1. In someembodiments, an IL4 variant, derivative, or fragment thereof does notcontain a K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F,Y124D, S125F, S128G, S125R, or S129A substitution.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof of the disclosure binds to an IL4 receptor subunit with about acomparable affinity as a wild type IL4 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold increasedaffinity compared to a wild type IL4 sequence. A comparable affinity canbe, for example, less than about 10, less than about 5, less than about2, less than about 2, less than about 1.9, less than about 1.8, lessthan about 1.7, less than about 1.6, less than about 1.5, less thanabout 1.4, less than about 1.3, less than about 1.2, or less than about1.1 fold decreased affinity compared to a wild type IL4 sequence.

For example, an IL4 or IL4 variant, derivative, or fragment thereof ofthe disclosure can bind to an interleukin 13 receptor alpha 1(IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα),or a combination thereof, e.g. with about a comparable affinity as awild type IL4. In some embodiments, an IL4 or IL4 variant, derivative,or fragment thereof of the disclosure can bind to IL-13Rα1 with about acomparable affinity as a wild type IL4. In some embodiments, an IL4 orIL4 variant, derivative, or fragment thereof of the disclosure can bindto common gamma chain with about a comparable affinity as a wild typeIL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof of the disclosure can bind to IL-4Rα with about a comparableaffinity as a wild type IL4 sequence. In some embodiments, an IL4 or IL4variant, derivative, or fragment thereof of the disclosure can bind toIL-13Rα1 and common gamma chain with about a comparable affinity as awild type IL4. In some embodiments, an IL4 or IL4 variant, derivative,or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rαwith about a comparable affinity as a wild type IL4. In someembodiments, an IL4 or IL4 variant, derivative, or fragment thereof ofthe disclosure can bind to common gamma chain and IL-4Rα with about acomparable affinity as a wild type IL4. In some embodiments, an IL4 orIL4 variant, derivative, or fragment thereof of the disclosure can bindto IL-13Rα1, common gamma chain, and IL-4Rα with about a comparableaffinity as a wild type IL4.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof of the disclosure binds to an IL4 receptor subunit with at leasta comparable affinity as a wild type IL4 sequence. For example, an IL4or IL4 variant, derivative, or fragment thereof of the disclosure canbind to an interleukin 13 receptor alpha 1 (IL-13Rα1), common gammachain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereofwith at least a comparable affinity as a wild type IL4. In someembodiments, an IL4 or IL4 variant, derivative, or fragment thereof ofthe disclosure can bind to IL-13Rα1 with at least a comparable affinityas a wild type IL4. In some embodiments, an IL4 or IL4 variant,derivative, or fragment thereof of the disclosure can bind to commongamma chain with at least a comparable affinity as a wild type IL4. Insome embodiments, an IL4 or IL4 variant, derivative, or fragment thereofof the disclosure can bind to IL-4Rα with at least a comparable affinityas a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant,derivative, or fragment thereof of the disclosure can bind to IL-13Rα1and common gamma chain with at least a comparable affinity as a wildtype IL4. In some embodiments, an IL4 or IL4 variant, derivative, orfragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα withat least a comparable affinity as a wild type IL4. In some embodiments,an IL4 or IL4 variant, derivative, or fragment thereof of the disclosurecan bind to common gamma chain and IL-4Rα with at least a comparableaffinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant,derivative, or fragment thereof of the disclosure can bind to IL-13Rα1,common gamma chain, and IL-4Rα with at least a comparable affinity as awild type IL4.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof of the disclosure binds to an IL4 receptor subunit with at mosta comparable affinity as a wild type IL4 sequence. For example, an IL4or IL4 variant, derivative, or fragment thereof of the disclosure canbind to an interleukin 13 receptor alpha 1 (IL-13Rα1), common gammachain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereofwith at most a comparable affinity as a wild type IL4. In someembodiments, an IL4 or IL4 variant, derivative, or fragment thereof ofthe disclosure can bind to IL-13Rα1 with at most a comparable affinityas a wild type IL4. In some embodiments, an IL4 or IL4 variant,derivative, or fragment thereof of the disclosure can bind to commongamma chain with at most a comparable affinity as a wild type IL4. Insome embodiments, an IL4 or IL4 variant, derivative, or fragment thereofof the disclosure can bind to IL-4Rα with at most a comparable affinityas a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant,derivative, or fragment thereof of the disclosure can bind to IL-13Rα1and common gamma chain with at most a comparable affinity as a wild typeIL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with at most acomparable affinity as a wild type IL4. In some embodiments, an IL4 orIL4 variant, derivative, or fragment thereof of the disclosure can bindto common gamma chain and IL-4Rα with at most a comparable affinity as awild type IL4. In some embodiments, an IL4 or IL4 variant, derivative,or fragment thereof of the disclosure can bind to IL-13Rα1, common gammachain, and IL-4Rα with at most a comparable affinity as a wild type IL4.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof can bind to an IL-13Rα1 with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL4 sequence. In some embodiments, an IL4 or IL4variant, derivative, or fragment thereof can bind to an IL-13Rα1 with atleast about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL4 sequence.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof can bind to a common gamma chain with at least about 1.5 fold, 2fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL4 sequence. In some embodiments, an IL4 or IL4variant, derivative, or fragment thereof can bind to a common gammachain with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000fold, or 10,000 fold decreased affinity relative to a wild type IL4sequence.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof can bind to an IL-4Rα with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL4 sequence. In some embodiments, an IL4 or IL4variant, derivative, or fragment thereof can bind to an IL-4Rα with atleast about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL4 sequence.

In some embodiments, an IL4 or IL4 variant, derivative, or fragmentthereof of the disclosure can activate a native IL4 receptor. A nativeIL4 receptor can be, for example, a receptor comprising an IL-13Rα1subunit and an IL-4Rα subunit, or a common gamma chain subunit and anIL-4Rα subunit. In some embodiments, an IL4 or IL4 variant, derivative,or fragment thereof of the disclosure can activate a native IL4 receptorwhen present in a fusion protein. In some embodiments, an IL4 or IL4variant, derivative, or fragment thereof of the disclosure can activatea native IL4 receptor when present as a polypeptide that is not part ofa fusion protein, but does not activate native IL4 receptor when presentin a fusion protein.

In some embodiments, a polypeptide of the disclosure does not containIL4. In some embodiments, a polypeptide of the disclosure does notcontain SEQ ID NO: 1.

Interleukin 10

A fusion protein may comprise an IL10 protein, or a variant, derivative,or fragment thereof operably linked or directly or indirectly fused toan interleukin 10 or a variant or derivative thereof. The IL10 proteinis preferably a mammalian IL10 protein, such as a human IL10, or mouseIL10. One amino acid sequence representing IL10 is set forth in SEQ IDNO:5. Variants of IL10 include, for example, proteins having at least70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as100%, amino acid sequence identity to SEQ ID NO:5, preferably over theentire length. Amino acid sequence identity is preferably determined bypairwise alignment using the Needleman and Wunsch algorithm and GAPdefault parameters as defined above. Variants, derivatives, andfragments thereof also include proteins having IL10 activity, which havebeen derived, by way of one or more amino acid substitutions, deletionsor insertions, from the polypeptide having the amino acid sequence ofSEQ ID NO:5. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30,25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL10 of the disclosure (e.g., an IL10 variant,derivative, or fragment thereof) can comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least or at least 50 amino acid substitutions, deletions, orinsertions relative to an IL10 sequence disclosed herein (e.g., a wildtype IL10 sequence).

In some embodiments, an IL10 of the disclosure (e.g., an IL10 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to an IL10sequence disclosed herein (e.g., a wild type IL10 sequence).

In some embodiments, an IL10 sequence of the disclosure (e.g., an IL10variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to an IL10 sequence disclosed herein(e.g., a wild type IL10 sequence).

In some embodiments, an IL10 sequence of the disclosure (e.g., an IL10variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to an IL10 sequencedisclosed herein (e.g., a wild type IL10 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

An IL10 of the disclosure can comprise a wild type IL10 sequence. Anon-limiting examples of a wild type IL10 sequences is SEQ ID NO: 5. SEQID NO: 5 can be a canonical wild type IL10 sequence of the disclosure.

An IL10 of the disclosure can comprise an IL10 variant, derivative, orfragment thereof with one or more amino acid substitutions. For example,an IL10 variant, derivative, or fragment thereof can comprise an aminoacid substitution at position 187, A89, H109, R110, F111,Y153, M156, ora combination thereof of SEQ ID NO: 5. In some embodiments, an IL10variant, derivative, or fragment thereof comprises a substitution thatis M156, F111S, I87A, I87G, A89D, HI09D, R110D, YI53D, MI56D, A89D,HI09E, R110E, YI53E, MI56E, or a combination thereof relative to SEQ IDNO: 5.

In some embodiments, an IL10 variant, derivative, or fragment thereofdoes not contain a substitution at position I87, A89, H109, R110,F111,Y153, or M156 relative to SEQ ID NO: 5. In some embodiments, anIL10 variant, derivative, or fragment thereof does not contain a M156,F111S, I87A, I87G, A89D, HI09D, R110D, YI53D, MI56D, A89D, HI09E, R110E,YI53E, or MI56E substitution.

In some embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure binds to an IL10 receptor subunit with about acomparable affinity as a wild type IL10 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 20 1.6, less than about 1.5, less than about 1.4, lessthan about 1.3, less than about 1.2, or less than about 1.1 foldincreased affinity compared to a wild type IL10 sequence. A comparableaffinity can be, for example, less than about 10, less than about 5,less than about 2, less than about 1.9, less than about 1.8, less thanabout 1.7, less than about 1.6, less than about 1.5, less than about1.4, less than about 1.3, less than about 1.2, or less than about 1.1fold decreased affinity compared to a wild type IL10 sequence.

For example, an IL10 or IL10 variant, derivative, or fragment thereof ofthe disclosure can bind to an interleukin 10 receptor 1 (IL-10R1),interleukin 10 receptor 2 (IL-10R2), or a combination thereof with abouta comparable affinity as a wild type IL10. In some embodiments, an IL10or IL10 variant, derivative, or fragment thereof of the disclosure canbind to IL-10R1 with about a comparable affinity as a wild type IL10. Insome embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure can bind to IL-10R2 with about a comparableaffinity as a wild type IL10. In some embodiments, an IL10 or IL10variant, derivative, or fragment thereof of the disclosure can bind toIL-10R1 and IL-10R2 with about a comparable affinity as a wild typeIL10.

In some embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure can bind to an IL10 receptor subunit with atleast a comparable affinity as a wild type IL10. For example, an IL10 orIL10 variant, derivative, or fragment thereof of the disclosure can bindto IL-10R1, IL-10R2, or a combination thereof with at least a comparableaffinity as a wild type IL10. In some embodiments, an IL10 or IL10variant, derivative, or fragment thereof of the disclosure can bind toan IL-10R1 with at least a comparable affinity as a wild type IL10. Insome embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure can bind to an IL-10R2 with at least acomparable affinity as a wild type IL10. In some embodiments, an IL10 orIL10 variant, derivative, or fragment thereof of the disclosure can bindto an IL-10R1 and an IL-10R2 with at least a comparable affinity as awild type IL10.

In some embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure can bind to an IL10 receptor subunit with atmost a comparable affinity as a wild type IL10. For example, an IL10 orIL10 variant, derivative, or fragment thereof of the disclosure can bindto IL-10R1, IL-10R2, or a combination thereof with at most a comparableaffinity as a wild type IL10. In some embodiments, an IL10 or IL10variant, derivative, or fragment thereof of the disclosure can bind toan IL-10R1 with at most a comparable affinity as a wild type IL10. Insome embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure can bind to an IL-10R2 with at most acomparable affinity as a wild type IL10. In some embodiments, an IL10 orIL10 variant, derivative, or fragment thereof of the disclosure can bindto an IL-10R1 and an IL-10R2 with at most a comparable affinity as awild type IL10.

In some embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof can bind to an IL-10R1 with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL10 sequence. In some embodiments, an IL10 orIL10 variant, derivative, or fragment thereof can bind to an IL-10R1with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL10 sequence.

In some embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof can bind to an IL-10R2 with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL10 sequence. In some embodiments, an IL10 orIL10 variant, derivative, or fragment thereof can bind to an IL-10R2with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL10 sequence.

In some embodiments, an IL10 or IL10 variant, derivative, or fragmentthereof of the disclosure can activate a native IL10 receptor. A nativeIL10 receptor can be, for example, a receptor comprising an IL-10R1subunit and an IL-10R2 subunit. In some embodiments, an IL10 or IL10variant, derivative, or fragment thereof of the disclosure can activatea native IL10 receptor when present in a fusion protein. In someembodiments, an IL10 or IL10 variant, derivative, or fragment thereof ofthe disclosure can activate a native IL10 receptor when present as apolypeptide that is not part of a fusion protein, but does not activatenative IL10 receptor when present in a fusion protein.

In some embodiments, a polypeptide of the disclosure does not containIL10. In some embodiments, a polypeptide of the disclosure does notcontain SEQ ID NO: 5.

Interleukin 33

A fusion protein may comprise an IL33 protein, or a variant, derivative,or fragment thereof operably linked or directly or indirectly fused toan interleukin 13 or a variant or derivative thereof. The IL33 proteinis preferably a mammalian IL33 protein, such as a human IL33, or mouseIL33. Non-limiting examples of amino acid sequences representing IL33include SEQ ID NO:6 and SEQ ID NOs: 29-34. Variants of IL33 include, forexample, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%,96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identityto SEQ ID NO:6 or any one of SEQ ID NOs: 29-34, preferably over theentire length. Amino acid sequence identity is preferably determined bypairwise alignment using the Needleman and Wunsch algorithm and GAPdefault parameters as defined above. Variants, derivatives, andfragments thereof also include proteins having IL33 activity, which havebeen derived, by way of one or more amino acid substitutions, deletionsor insertions, from the polypeptide having the amino acid sequence ofSEQ ID NO:6 or any one of SEQ ID NOs: 29-34. Preferably, such proteinscomprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90,80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions,deletions or insertions.

In some embodiments, an IL33 of the disclosure (e.g., an IL33 variant,derivative, or fragment thereof) can comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least or at least 50 amino acid substitutions, deletions, orinsertions relative to an IL33 sequence disclosed herein (e.g., a wildtype IL33 sequence).

In some embodiments, an IL33 of the disclosure (e.g., an IL33 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to an IL33sequence disclosed herein (e.g., a wild type IL33 sequence).

In some embodiments, an IL33 sequence of the disclosure (e.g., an IL33variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to an IL33 sequence disclosed herein(e.g., a wild type IL33 sequence).

In some embodiments, an IL33 sequence of the disclosure (e.g., an IL33variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to an IL33 sequencedisclosed herein (e.g., a wild type IL33 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

An IL33 of the disclosure can comprise a wild type IL33 sequence.Non-limiting examples of wild type IL33 sequences include SEQ ID NOs: 6and 29-34. SEQ ID NO: 6 can be a canonical wild type IL33 sequence.

An IL33 of the disclosure can comprise an IL33 variant, derivative, orfragment thereof with one or more amino acid substitutions. For example,an IL33 variant, derivative, or fragment thereof can comprise an aminoacid substitution at position I263 of SEQ ID NO: 6. In some embodiments,an IL33 variant, derivative, or fragment thereof comprises asubstitution that is I263M relative to SEQ ID NO: 6.

In some embodiments, an IL33 variant, derivative, or fragment thereofdoes not contain a substitution at position I263 relative to SEQ ID NO:6. In some embodiments, an IL33 variant, derivative, or fragment thereofdoes not contain an I263M substitution.

In some embodiments, an IL33 or IL33 variant, derivative, or fragmentthereof of the disclosure binds to an IL33 receptor subunit with about acomparable affinity as a wild type IL33 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold increasedaffinity compared to a wild type IL33 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold decreasedaffinity compared to a wild type IL33 sequence.

For example, an IL33 or IL33 variant, derivative, or fragment thereof ofthe disclosure can bind to ST2 (IL1 RL1), IL1RAP, or a combinationthereof with about a comparable affinity as a wild type IL33. In someembodiments, an IL33 or IL33 variant, derivative, or fragment thereof ofthe disclosure can bind to ST2 with about a comparable affinity as awild type IL33. In some embodiments, an IL33 or IL33 variant,derivative, or fragment thereof of the disclosure can bind to IL1RAPwith about a comparable affinity as a wild type IL33. In someembodiments, an IL33 or IL33 variant, derivative, or fragment thereof ofthe disclosure can bind to ST2 and IL1RAP with about a comparableaffinity as a wild type IL33.

In some embodiments, an IL33 or IL33 variant, derivative, or fragmentthereof of the disclosure can bind to an IL33 receptor subunit with atleast a comparable affinity as a wild type IL33. For example, an IL33 orIL33 variant, derivative, or fragment thereof of the disclosure can bindto ST2, IL1RAP, or a combination thereof with at least a comparableaffinity as a wild type IL33. In some embodiments, an IL33 or IL33variant, derivative, or fragment thereof of the disclosure can bind toan ST2 with at least a comparable affinity as a wild type IL33. In someembodiments, an IL33 or IL33 variant, derivative, or fragment thereof ofthe disclosure can bind to an IL1RAP with at least a comparable affinityas a wild type IL33. In some embodiments, an IL33 or IL33 variant,derivative, or fragment thereof of the disclosure can bind to an ST2 andan IL1RAP with at least a comparable affinity as a wild type IL33.

In some embodiments, an IL33 or IL33 variant, derivative, or fragmentthereof of the disclosure can bind to an IL33 receptor subunit with atmost a comparable affinity as a wild type IL33. For example, an IL33 orIL33 variant, derivative, or fragment thereof of the disclosure can bindto ST2, IL1RAP, or a combination thereof with at most a comparableaffinity as a wild type IL33. In some embodiments, an IL33 or IL33variant, derivative, or fragment thereof of the disclosure can bind toan ST2 with at most a comparable affinity as a wild type IL33. In someembodiments, an IL33 or IL33 variant, derivative, or fragment thereof ofthe disclosure can bind to an IL1RAP with at most a comparable affinityas a wild type IL33. In some embodiments, an IL33 or IL33 variant,derivative, or fragment thereof of the disclosure can bind to an ST2 andan IL1RAP with at most a comparable affinity as a wild type IL33.

In some embodiments, an IL33 or IL33 variant, derivative, or fragmentthereof can bind to an ST2 with at least about 1.5 fold, 2 fold, 5 fold,10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative toa wild type IL33 sequence. In some embodiments, an IL33 or IL33 variant,derivative, or fragment thereof can bind to an ST2 with at least about1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold,50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 folddecreased affinity relative to a wild type IL33 sequence.

In some embodiments, an IL33 or IL33 variant, derivative, or fragmentthereof can bind to an IL1RAP with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL33 sequence. In some embodiments, an IL33 orIL33 variant, derivative, or fragment thereof can bind to an IL1RAP withat least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL33 sequence.

In some embodiments, an IL33 or IL33 variant, derivative, or fragmentthereof of the disclosure can activate a native IL33 receptor. A nativeIL33 receptor can be, for example, a receptor comprising an ST2 subunitand an IL1RAP subunit. In some embodiments, an IL33 or IL33 variant,derivative, or fragment thereof of the disclosure can activate a nativeIL33 receptor when present in a fusion protein. In some embodiments, anIL33 or IL33 variant, derivative, or fragment thereof of the disclosurecan activate a native IL33 receptor when present as a polypeptide thatis not part of a fusion protein, but does not activate native IL33receptor when present in a fusion protein.

Transforming Growth Factor Beta 1

A fusion protein may comprise a TGFβ1 protein, or a variant, derivative,or fragment thereof operably linked or directly or indirectly fused toan interleukin 13 or a variant or derivative thereof. The TGFβ1 proteinis preferably a mammalian TGFβ1 protein, such as a human TGFβ1, or mouseTGFβ1. Non-limiting examples of amino acid sequences representing TGFβ1include SEQ ID NO:7 and SEQ ID NO: 21. Variants of TGFβ1 include, forexample, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%,96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identityto SEQ ID NO:7 (whole or underlined part) or SEQ ID NO: 21, preferablyover the entire length. Amino acid sequence identity is preferablydetermined by pairwise alignment using the Needleman and Wunschalgorithm and GAP default parameters as defined above. Variants,derivatives, or fragments thereof also include proteins having TGFβ1activity, which have been derived, by way of one or more amino acidsubstitutions, deletions or insertions, from the polypeptide having theamino acid sequence of SEQ ID NO:7 or SEQ ID NO: 21. Preferably, suchproteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acidsubstitutions, deletions or insertions.

In some embodiments, a TGFβ1 of the disclosure (e.g., a TGFβ1 variant,derivative, or fragment thereof) can comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least or at least 50 amino acid substitutions, deletions, orinsertions relative to a TGFβ1 sequence disclosed herein (e.g., a wildtype TGFβ1 sequence).

In some embodiments, a TGFβ1 of the disclosure (e.g., a TGFβ1 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to a TGFβ1sequence disclosed herein (e.g., a wild type TGFβ1 sequence).

In some embodiments, a TGFβ1 sequence of the disclosure (e.g., a TGFβ1variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to a TGFβ1 sequence disclosed herein(e.g., a wild type TGFβ1 sequence).

In some embodiments, a TGFβ1 sequence of the disclosure (e.g., a TGFβ1variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to a TGFβ1 sequence 10disclosed herein (e.g., a wild type TGFβ1 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

A TGFβ1 of the disclosure can comprise a wild type TGFβ1 sequence.Non-limiting examples of wild type TGFβ1 sequences include SEQ ID NOs: 7and 21. A canonical TGFβ1 sequence can be SEQ ID NO: 21.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure binds to a TGFβ1 receptor subunit with about acomparable affinity as a wild type TGFβ1 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold increasedaffinity compared to a wild type TGFβ1 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold decreasedaffinity compared to a wild type TGFβ1 sequence.

For example, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereofof the disclosure can bind to a transforming growth factor beta receptor1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), anactivin receptor-like kinase 1 (ALK-1), an activin receptor-like kinase2 (ALK-2), or a combination thereof with about a comparable affinity asa wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant,derivative, or fragment thereof of the disclosure can bind to TGFβR1with about a comparable affinity as a wild type TGFβ1. In someembodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereofof the disclosure can bind to TGFβR2 with about a comparable affinity asa wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant,derivative, or fragment thereof of the disclosure can bind to ALK-1 withabout a comparable affinity as a wild type TGFβ1 sequence. In someembodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereofof the disclosure can bind to ALK-2 with about a comparable affinity asa wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1variant, derivative, or fragment thereof of the disclosure can bind to aTGFβR1, TGFβR2, ALK-1, and ALK-2 with about a comparable affinity as awild type TGFβ1 sequence.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can bind to a transforming growth factor betareceptor 1 (TGFβR1), a transforming growth factor beta receptor 2(TGFβR2), an activin receptor-like kinase 1 (ALK-1), an activinreceptor-like kinase 2 (ALK-2), or a combination thereof with at least acomparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1or TGFβ1 variant, derivative, or fragment thereof of the disclosure canbind to TGFβR1 with at least a comparable affinity as a wild type TGFβ1.In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can bind to TGFβR2 with at least a comparableaffinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1variant, derivative, or fragment thereof of the disclosure can bind toALK-1 with at least a comparable affinity as a wild type TGFβ1 sequence.In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can bind to ALK-2 with at least a comparableaffinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 orTGFβ1 variant, derivative, or fragment thereof of the disclosure canbind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at least a comparableaffinity as a wild type TGFβ1 sequence.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can bind to a transforming growth factor betareceptor 1 (TGFβR1), a transforming growth factor beta receptor 2(TGFβR2), an activin receptor-like kinase 1 (ALK-1), an activinreceptor-like kinase 2 (ALK-2), or a combination thereof with at most acomparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1or TGFβ1 variant, derivative, or fragment thereof of the disclosure canbind to TGFβR1 with at most a comparable affinity as a wild type TGFβ1.In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can bind to TGFβR2 with at most a comparableaffinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1variant, derivative, or fragment thereof of the disclosure can bind toALK-1 with at most a comparable affinity as a wild type TGFβ1 sequence.In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can bind to ALK-2 with at most a comparableaffinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 orTGFβ1 variant, derivative, or fragment thereof of the disclosure canbind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at most a comparableaffinity as a wild type TGFβ1 sequence.

In some embodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof can bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at leastabout 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 foldincreased affinity relative to a wild type TGFβ1 sequence. In someembodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragment thereofcan bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreasedaffinity relative to a wild type TGFβ1 sequence.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragmentthereof of the disclosure can activate a native TGFβ1 receptor. A nativeTGFβ1 receptor can be, for example, a receptor comprising a TGFβR1subunit and a TGFβR2 subunit. In some embodiments, an TGFβ1 or TGFβ1variant, derivative, or fragment thereof of the disclosure can activatea native TGFβ1 receptor when present in a fusion protein. In someembodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragment thereofof the disclosure can activate a native TGFβ1 receptor when present as apolypeptide that is not part of a fusion protein, but does not activatenative TGFβ1 receptor when present in a fusion protein.

Transforming Growth Factor Beta 2

A fusion protein may comprise a TGFβ2 protein, or a variant, derivative,or fragment thereof operably linked or directly or indirectly fused toan interleukin 13 or a variant or derivative thereof. The TGFβ2 proteinis preferably a mammalian TGFβ2 protein, such as a human TGFβ2, or mouseTGFβ2. Non-limiting examples of amino acid sequences representing TGFβ2include SEQ ID NO:8, SEQ ID NO: 22, and SEQ ID NO: 35. Variants of TGFβ2include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%,92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequenceidentity to SEQ ID NO:8 (whole or underlined part), SEQ ID NO: 22, orSEQ ID NO: 35, preferably over the entire length. Amino acid sequenceidentity is preferably determined by pairwise alignment using theNeedleman and Wunsch algorithm and GAP default parameters as definedabove. Variants, derivatives, and fragments thereof also includeproteins having TGFβ2 activity, which have been derived, by way of oneor more amino acid substitutions, deletions or insertions, from thepolypeptide having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:22, or SEQ ID NO: 35. Preferably, such proteins comprise from 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45,40, 35, 30, 25, 20, 15 amino acid substitutions, deletions orinsertions.

In some embodiments, a TGFβ2 of the disclosure (e.g., a TGFβ2 variant,derivative, or fragment thereof) can comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least or at least 50 amino acid substitutions, deletions, orinsertions relative to a TGFβ2 sequence disclosed herein (e.g., a wildtype TGFβ2 sequence).

In some embodiments, a TGFβ2 of the disclosure (e.g., a TGFβ2 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to a TGFβ2sequence disclosed herein (e.g., a wild type TGFβ2 sequence).

In some embodiments, a TGFβ2 sequence of the disclosure (e.g., a TGFβ2variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to a TGFβ2 sequence disclosed herein(e.g., a wild type TGFβ2 sequence).

In some embodiments, a TGFβ2 sequence of the disclosure (e.g., a TGFβ2variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to a TGFβ2 sequencedisclosed herein (e.g., a wild type TGFβ2 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

A TGFβ2 of the disclosure can comprise a wild type TGFβ2 sequence.Non-limiting examples of wild type TGFβ2 sequences include SEQ ID NOs:8, 22, and 35. SEQ ID NO: 22 can be a canonical wild type TGFβ2 sequenceof the disclosure.

A TGFβ2 of the disclosure can comprise an TGFβ2 variant, derivative, orfragment thereof with one or more amino acid substitutions. For example,a TGFβ2 variant, derivative, or fragment thereof can comprise an aminoacid substitution at position R18, P36, or a combination thereof of SEQID NO: 22. In some embodiments, a TGFβ2 variant, derivative, or fragmentthereof comprises a substitution that is R18C, P36H, or a combinationthereof relative to SEQ ID NO: 22. In some embodiments, a TGFβ2,fragment, or derivative thereof comprises the substitutions R18C, andP36H relative to SEQ ID NO: 22.

In some embodiments, a TGFβ2 variant, derivative, or fragment thereofdoes not contain a substitution at position R18, or P36 relative to SEQID NO: 22. In some embodiments, a TGFβ2 variant, derivative, or fragmentthereof does not contain a R18C or P36H substitution.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure binds to a TGFβ2 receptor subunit with about acomparable affinity as a wild type TGFβ2 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold increasedaffinity compared to a wild type TGFβ2 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold decreasedaffinity compared to a wild type TGFβ2 sequence.

For example, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereofof the disclosure can bind to a transforming growth factor beta receptor1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), anactivin receptor-like kinase 1 (ALK-1), an activin receptor-like kinase2 (ALK-2), or a combination thereof with about a comparable affinity asa wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant,derivative, or fragment thereof of the disclosure can bind to TGFβR1with about a comparable affinity as a wild type TGFβ2. In someembodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereofof the disclosure can bind to TGFβR2 with about a comparable affinity asa wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant,derivative, or fragment thereof of the disclosure can bind to ALK-1 withabout a comparable affinity as a wild type TGFβ2 sequence. In someembodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereofof the disclosure can bind to ALK-2 with about a comparable affinity asa wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2variant, derivative, or fragment thereof of the disclosure can bind to aTGFβR1, TGFβR2, ALK-1, and ALK-2 with about a comparable affinity as awild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can bind to a transforming growth factor betareceptor 1 (TGFβR1), a transforming growth factor beta receptor 2(TGFβR2), an activin receptor-like kinase 1 (ALK-1), an activinreceptor-like kinase 2 (ALK-2), or a combination thereof with at least acomparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2or TGFβ2 variant, derivative, or fragment thereof of the disclosure canbind to TGFβR1 with at least a comparable affinity as a wild type TGFβ2.In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can bind to TGFβR2 with at least a comparableaffinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2variant, derivative, or fragment thereof of the disclosure can bind toALK-1 with at least a comparable affinity as a wild type TGFβ2 sequence.In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can bind to ALK-2 with at least a comparableaffinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 orTGFβ2 variant, derivative, or fragment thereof of the disclosure canbind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at least a comparableaffinity as a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can bind to a transforming growth factor betareceptor 1 (TGFβR1), a transforming growth factor beta receptor 2(TGFβR2), an activin receptor-like kinase 1 (ALK-1), an activinreceptor-like kinase 2 (ALK-2), or a combination thereof with at most acomparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2or TGFβ2 variant, derivative, or fragment thereof of the disclosure canbind to TGFβR1 with at most a comparable affinity as a wild type TGFβ2.In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can bind to TGFβR2 with at most a comparableaffinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2variant, derivative, or fragment thereof of the disclosure can bind toALK-1 with at most a comparable affinity as a wild type TGFβ2 sequence.In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can bind to ALK-2 with at most a comparableaffinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 orTGFβ2 variant, derivative, or fragment thereof of the disclosure canbind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at most a comparableaffinity as a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof can bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at leastabout 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 foldincreased affinity relative to a wild type TGFβ2 sequence. In someembodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereofcan bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreasedaffinity relative to a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragmentthereof of the disclosure can activate a native TGFβ2 receptor. A nativeTGFβ2 receptor can be, for example, a receptor comprising a TGFβR1subunit and a TGFβR2 subunit. In some embodiments, an TGFβ2 or TGFβ2variant, derivative, or fragment thereof of the disclosure can activatea native TGFβ2 receptor when present in a fusion protein. In someembodiments, an TGFβ2 or TGFβ2 variant, derivative, or fragment thereofof the disclosure can activate a native TGFβ2 receptor when present as apolypeptide that is not part of a fusion protein, but does not activatenative TGFβ2 receptor when present in a fusion protein.

Interleukin 27

A fusion protein may comprise an IL27 protein, or a variant, derivative,or fragment thereof operably linked or directly or indirectly fused toan interleukin 13 or a variant or derivative thereof. The IL27 proteinis preferably a mammalian IL27 protein, such as a human IL27, or mouseIL27, or a variant, derivative, or fragment thereof. An IL27 or an IL27variant, derivative, or fragment thereof of the disclosure can comprisean IL27A subunit, an IL27B (EBI3) subunit, or a combination thereof. Insome embodiments, an IL27 of the disclosure comprises an IL27A subunit.In some embodiments, an IL27 of the disclosure comprises a variant IL27Asubunit as disclosed below (e.g., as provided in SEQ ID NO: 18). In someembodiments, an IL27 of the disclosure comprises an IL27B subunit.

An example of an amino acid sequence representing IL27A is set forth inSEQ ID NO:36. An example of an amino acid sequence representing IL27B isset forth in SEQ ID NO:45. Variants of IL27 include, for example,proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%,98%, 99% or more, such as 100%, amino acid sequence identity to SEQ IDNO:36 or SEQ ID NO: 45, preferably over the entire length. Amino acidsequence identity is preferably determined by pairwise alignment usingthe Needleman and Wunsch algorithm and GAP default parameters as definedabove. Variants, derivatives, and fragments thereof also includeproteins having IL27 activity, which have been derived, by way of one ormore amino acid substitutions, deletions or insertions, from thepolypeptide having the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:45. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20,15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL27 of the disclosure (e.g., an IL27 variant,derivative, or fragment thereof) can comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least or at least 50 amino acid substitutions, deletions, orinsertions relative to an IL27 sequence disclosed herein (e.g., a wildtype IL27 sequence).

In some embodiments, an IL27 of the disclosure (e.g., an IL27 variant,derivative, or fragment thereof) can comprise at most 1, at most 2, atmost 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, at most 12, at most 13, at most 14, at most15, at most 16, at most 17, at most 18, at most 19, at most 20, at most25, at most 30, at most 35, at most 40, at most 45, or at most 50 aminoacid substitutions, deletions, or insertions relative to an IL27sequence disclosed herein (e.g., a wild type IL27 sequence).

In some embodiments, an IL27 sequence of the disclosure (e.g., an IL27variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions,deletions, or insertions relative to an IL27 sequence disclosed herein(e.g., a wild type IL27 sequence).

In some embodiments, an IL27 sequence of the disclosure (e.g., an IL27variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions, deletions, or insertions relative to an IL27 sequencedisclosed herein (e.g., a wild type IL27 sequence). An amino acidsubstitution can be a conservative or a non-conservative substitution.The one or more amino acid substitutions, deletions, or insertions canbe at the N-terminus, the C-terminus, within the amino acid sequence, ora combination thereof. The amino acid substitutions, deletions, orinsertions can be contiguous, non-contiguous, or a combination thereof.

An IL27 of the disclosure can comprise a wild type IL27 sequence.Non-limiting examples of wild type IL27 sequences include SEQ ID NO: 36(IL27A) and SEQ ID NO: 45 (IL27B). SEQ ID NO: 36 can be a canonical wildtype IL27 sequence. In some embodiments, an IL27 sequence of thedisclosure comprises one substitution relative to a wild type IL27sequence.

An example of an IL27 variant, derivative, or fragment thereof of thedisclosure is an IL27 variant sequence that can be secreted as afunctional immune modulatory monomer protein, for example, an IL27Avariant, derivative, or fragment thereof that can be secreted andfunction as a functional immune modulatory monomer protein withoutneeding to associate with an IL27B (EBI3) subunit. One or more aminoacid substitutions, deletions, or insertions can be introduced togenerate such a molecule. SEQ ID NO: 18 is an example of an IL27variant, derivative, or fragment thereof of the disclosure thatcomprises one amino acid substation L134C relative to SEQ ID NO: 36(which is L162C in the sequence that includes the signal peptide), andcan be secreted as a functional immune modulatory monomer protein.

An IL27 of the disclosure can comprise an IL27 variant, derivative, orfragment thereof with one or more amino acid substitutions. For example,an IL27 variant, derivative, or fragment thereof can comprise an aminoacid substitution at position F132, N132, L134, P135, E136, E137, L152,L153, P154, or a combination thereof of SEQ ID NO: 36. In someembodiments, an IL27 variant, derivative, or fragment thereof comprisesa substitution that is F132C, N132C, L134C, P135C, E136C, E137C, L152C,L153C, P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D,P154D, F132E, N132E, L134E, P135E, E136E, E137E, L152E, L153E, P154E,F132R, N132R, L134R, P135R, E136R, E137R, L152R, L153R, P154R, F132K,N132K, L134K, P135K, E136K, E137K, L152K, L153K, P154K, S31A, L91P, or acombination thereof relative to SEQ ID NO: 36.

In some embodiments, an IL27 variant, derivative, or fragment thereofdoes not contain a substitution at position F132, N132, L134, P135,E136, E137, L152, L153, or P154 relative to SEQ ID NO: 36. In someembodiments, an IL27 variant, derivative, or fragment thereof does notcontain an F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C,P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D, P154D,F132E, N132E, L134E, P135E, E136E, E137E, L152E, L153E, P154E, F132R,N132R, L134R, P135R, E136R, E137R, L152R, L153R, P154R, F132K, N132K,L134K, P135K, E136K, E137K, L152K, L153K, P154K, S31A, or L91Psubstitution.

In some embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof of the disclosure binds to an IL27 receptor subunit with about acomparable affinity as a wild type IL27 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold increasedaffinity compared to a wild type IL27 sequence. A comparable affinitycan be, for example, less than about 10, less than about 5, less thanabout 2, less than about 1.9, less than about 1.8, less than about 1.7,less than about 1.6, less than about 1.5, less than about 1.4, less thanabout 1.3, less than about 1.2, or less than about 1.1 fold decreasedaffinity compared to a wild type IL27 sequence.

For example, an IL27 or IL27 variant, derivative, or fragment thereof ofthe disclosure can bind to an interleukin 27 receptor alpha (IL-27RA),gp130, or a combination thereof with about a comparable affinity as awild type IL27. In some embodiments, an IL27 or IL27 variant,derivative, or fragment thereof of the disclosure can bind to IL-27RAwith about a comparable affinity as a wild type IL27. In someembodiments, an IL27 or IL27 variant, derivative, or fragment thereof ofthe disclosure can bind to gp130 with about a comparable affinity as awild type IL27. In some embodiments, an IL27 or IL27 variant,derivative, or fragment thereof of the disclosure can bind to IL-27RAand gp130 with about a comparable affinity as a wild type IL27.

In some embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof of the disclosure can bind to an IL27 receptor subunit with atleast a comparable affinity as a wild type IL27. For example, an IL27 orIL27 variant, derivative, or fragment thereof of the disclosure can bindto IL-27RA, gp130, or a combination thereof with at least a comparableaffinity as a wild type IL27. In some embodiments, an IL27 or IL27variant, derivative, or fragment thereof of the disclosure can bind toan IL-27RA with at least a comparable affinity as a wild type IL27. Insome embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof of the disclosure can bind to an gp130 with at least acomparable affinity as a wild type IL27. In some embodiments, an IL27 orIL27 variant, derivative, or fragment thereof of the disclosure can bindto an IL-27RA and an gp130 with at least a comparable affinity as a wildtype IL27.

In some embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof of the disclosure can bind to an IL27 receptor subunit with atmost a comparable affinity as a wild type IL27. For example, an IL27 orIL27 variant, derivative, or fragment thereof of the disclosure can bindto IL-27RA, gp130, or a combination thereof with at most a comparableaffinity as a wild type IL27. In some embodiments, an IL27 or IL27variant, derivative, or fragment thereof of the disclosure can bind toan IL-27RA with at most a comparable affinity as a wild type IL27. Insome embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof of the disclosure can bind to an gp130 with at most a comparableaffinity as a wild type IL27. In some embodiments, an IL27 or IL27variant, derivative, or fragment thereof of the disclosure can bind toan IL-27RA and an gp130 with at most a comparable affinity as a wildtype IL27.

In some embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof can bind to an IL-27RA with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL27 sequence. In some embodiments, an IL27 orIL27 variant, derivative, or fragment thereof can bind to an IL-27RAwith at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL27 sequence.

In some embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof can bind to gp130 A with at least about 1.5 fold, 2 fold, 5fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinityrelative to a wild type IL27 sequence. In some embodiments, an IL27 orIL27 variant, derivative, or fragment thereof can bind to gp130 with atleast about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or10,000 fold decreased affinity relative to a wild type IL27 sequence.

In some embodiments, an IL27 or IL27 variant, derivative, or fragmentthereof of the disclosure can activate a native IL27 receptor. A nativeIL27 receptor can be, for example, a receptor comprising an IL-27RAsubunit and a gp130 subunit. In some embodiments, an IL27 or IL27variant, derivative, or fragment thereof of the disclosure can activatea native IL27 receptor when present in a fusion protein. In someembodiments, an IL27 or IL27 variant, derivative, or fragment thereof ofthe disclosure can activate a native IL27 receptor when present as apolypeptide that is not part of a fusion protein, but does not activatenative IL27 receptor when present in a fusion protein.

Fusion Proteins

The present disclosure provides fusion proteins that comprise aninterleukin 13 (IL13) directly or indirectly linked to a regulatorycytokine, for example, IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or anotherIL13.

In some embodiments, a fusion protein of the disclosure can bind toreceptors present on the surface of a cell and form a complex with about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, or about 12 receptor subunits (e.g., polypeptide chains).In some embodiments, a fusion protein of the disclosure can bind toreceptors present on the surface of a cell and form a complex with atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, or at least 12 receptorsubunits (e.g., polypeptide chains). In some embodiments, a fusionprotein of the disclosure can bind to receptors present on the surfaceof a cell and form a complex with at most 2, at most 3, at most 4, atmost 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most11, or at most 12 receptor subunits (e.g., polypeptide chains).

The IL4 (or IL10 or IL13 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13 inthe fusion protein may or may not be connected by a linker, e.g., alinker sequence, or by a chemical spacer.

A linker can be a peptide. A linker can comprise a linker sequence, forexample, a linker peptide sequence. A linker sequence can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 51, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 aminoacid residues in length.

A linker as described herein can include a flexible or rigid linker. Aflexible linker can have a sequence containing stretches of glycine andserine residues. The small size of the glycine and serine residuesprovides flexibility, and allows for mobility of the connectedfunctional domains. The incorporation of serine or threonine canmaintain the stability of the linker in aqueous solutions by forminghydrogen bonds with the water molecules, thereby reducing unfavorableinteractions between the linker and protein moieties. Flexible linkerscan also contain additional amino acids such as threonine and alanine tomaintain flexibility, as well as polar amino acids such as lysine andglutamine to improve solubility.

A flexible linker can comprise SEQ ID NO: 3. A flexible linker cancomprise repeats of SEQ ID NO: 37 (GGGS), for example, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 repeats of SEQ ID NO: 37. A flexible linker can compriserepeats of SEQ ID NO: 38 (GGGGS), for example, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 repeats of SEQ ID NO: 38. Several other types of flexiblelinkers, including SEQ ID NO: 40 (KESGSVSSEQLAQFRSLD) and SEQ ID NO: 41(EGKSSGSGSESKST), can also be used. The SEQ ID NO: 42 (GSAGSAAGSGEF)linker can also be used, in which large hydrophobic residues areminimized to maintain good solubility in aqueous solutions. The lengthof the flexible linkers can be adjusted to allow for proper folding orto achieve optimal biological activity of the fused proteins.

A rigid linker can have, for example, an alpha helix-structure. Analpha-helical rigid linker can act as a spacer between protein domains.A rigid linker can comprise repeats of SEQ ID NO: 43 (EAAAK), forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 43. Arigid linker can comprise repeats of SEQ ID NO: 44 (EAAAR), for example,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 44. A rigidlinker can have a proline-rich sequence, (XP)n, with X designatingalanine, lysine, glutamine, or any amino acid. The presence of prolinein non-helical linkers can increase stiffness, and allow for effectiveseparation of protein domains.

A linker of the disclosure can include a non-peptide linker, forexample, a chemical linker. For example, two amino acid sequences of thedisclosure can be connected by a chemical linker. Each chemical linkerof the disclosure can be alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, any of which is optionally substituted. In someembodiments, a chemical linker of the disclosure can be an ester, ether,amide, thioether, or polyethyleneglycol (PEG). In some embodiments, alinker can reverse the order of the amino acids sequence in a compound,for example, so that the amino acid sequences linked by the linked arehead-to-head, rather than head-to-tail. Non-limiting examples of suchlinkers include diesters of dicarboxylic acids, such as oxalyl diester,malonyl diester, succinyl diester, glutaryl diester, adipyl diester,pimetyl diester, fumaryl diester, maleyl diester, phthalyl diester,isophthalyl diester, and terephthalyl diester. Non-limiting examples ofsuch linkers include diamides of dicarboxylic acids, such as oxalyldiamide, malonyl diamide, succinyl diamide, glutaryl diamide, adipyldiamide, pimetyl diamide, fumaryl diamide, maleyl diamide, phthalyldiamide, isophthalyl diamide, and terephthalyl diamide. Non-limitingexamples of such linkers include diamides of diamino linkers, such asethylene diamine, 1,2-di(methylamino)ethane, 1,3-diaminopropane,1,3-di(methylamino)propane, 1,4-di(methylamino)butane,1,5-di(methylamino)pentane, 1,6-di(methylamino)hexane, and pipyrizine.Non-limiting examples of optional substituents include hydroxyl groups,sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups,cyano groups, azido groups, sulfoxide groups, sulfone groups,sulfonamide groups, carboxyl groups, carboxaldehyde groups, iminegroups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenylgroups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups,aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups,acyl groups, acyloxy groups, carbamate groups, amide groups, ureidogroups, epoxy groups, and ester groups.

In some embodiments, an N terminus of a polypeptide sequence can belinked to an N terminus of another polypeptide. In some embodiments, a Cterminus of a polypeptide sequence can be linked to a C terminus ofanother polypeptide.

In some embodiments, a fusion protein of the disclosure does not containa linker.

Amino acids can include genetically encoded and non-genetically encodedoccurring amino acids. Amino acids can include naturally occurring andnon-naturally occurring amino acids. Amino acids can be L forms or Dforms. Substitutions can include conservative and/or non-conservativeamino acid substitutions. A conservative amino acid substitution can bea substitution of one amino acid for another amino acid of similarbiochemical properties (e.g., charge, size, and/or hydrophobicity). Anon-conservative amino acid substitution can be a substitution of oneamino acid for another amino acid with different biochemical properties(e.g., charge, size, and/or hydrophobicity). A conservative amino acidchange can be, for example, substitution that has minimal effect on thesecondary or tertiary structure of a polypeptide. A conservative aminoacid change can be an amino acid change from one hydrophilic amino acidto another hydrophilic amino acid. Hydrophilic amino acids can includeThr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K)and Arg (R). A conservative amino acid change can be an amino acidchange from one hydrophobic amino acid to another hydrophilic aminoacid. Hydrophobic amino acids can include Ile (I), Phe (F), Val (V), Leu(L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). Aconservative amino acid change can be an amino acid change from oneacidic amino acid to another acidic amino acid. Acidic amino acids caninclude Glu (E) and Asp (D). A conservative amino acid change can be anamino acid change from one basic amino acid to another basic amino acid.Basic amino acids can include His (H), Arg (R) and Lys (K). Aconservative amino acid change can be an amino acid change from onepolar amino acid to another polar amino acid. Polar amino acids caninclude Asn (N), Gln (Q), Ser (S) and Thr (T). A conservative amino acidchange can be an amino acid change from one nonpolar amino acid toanother nonpolar amino acid. Nonpolar amino acids can include Leu (L),Val(V), Ile (I), Met (M), Gly (G) and Ala (A). A conservative amino acidchange can be an amino acid change from one aromatic amino acid toanother aromatic amino acid. Aromatic amino acids can include Phe (F),Tyr (Y) and Trp (W). A conservative amino acid change can be an aminoacid change from one aliphatic amino acid to another aliphatic aminoacid. Aliphatic amino acids can include Ala (A), Val (V), Leu (L) andIle (I). In some embodiments, a conservative amino acid substitution isan amino acid change from one amino acid to another amino acid withinone of the following groups: Group I: ala, pro, gly, gln, asn, ser, thr;Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe;Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp,glu.

Additional amino acid sequences may present at the N- and/or C-terminusof the fusion protein of the present invention, e.g., an affinity tag tofacilitate purification. For example, a poly-histidine-tag, GST-tag,FLAG-tag, CBP tag, HA tag, or Myc tag may be present at the C- orN-terminus to facilitate purification. In some embodiments, an affinitytag is removed from a fusion protein of the disclosure, e.g., afterpurification. In some embodiments, a fusion protein of the disclosuredoes not contain an affinity tag, (e.g., the fusion protein can bepurified by other methods). Additionally or alternatively, the fusionprotein of the invention may optionally comprise additional proteinmoieties, such as moieties capable of targeting, e.g., a protein moietycomprising one or more antibody Fc regions. In some embodiments, afusion protein comprises an antibody Fc region. In some embodiments, afusion protein comprises an extracellular matrix-binding polypeptide.

The IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) may be locatedN-terminal of the IL13, or may be located C-terminal of the IL13. In apreferred embodiment, the IL4 (or IL10 or IL27 or IL33 or TGFβ1 orTGFβ2) molecule is located N-terminal of the IL13 molecule.

In an embodiment, the fusion protein of the invention consistsessentially of IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13,optionally linked by a linker sequence.

In an embodiment, the fusion protein of the present invention preventsor reduces neuronal damage to primary sensory neurons cultured overnightin presence of oxaliplatin or paclitaxel as quantified by measuring theneurite length after β3-tubulin staining.

In a suitable embodiment, the fusion protein of the present invention ispresent in, purified into, and/or used in a monomeric form. In oneembodiment, it has a molecular weight of 30 to 37 kDa. In someembodiments, a fusion protein of the disclosure is present in, purifiedinto, and/or used in a multimeric form, for example, a dimeric form or atetrameric form. In some embodiments, a fusion protein of the disclosureis present as, purified into, and/or used as a monomer, a dimer, atrimer, a tetramer, a multimer, or any combination thereof. A dimer,trimer, tetramer, or multimer can comprise subunits that are covalentlyor non-covalently bound.

In some embodiments, an IL4/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as a monomer. In someembodiments, an IL4/IL13 fusion protein of the disclosure is present as,purified into, and/or used as a dimer. In some embodiments, an IL4/IL13fusion protein of the disclosure is present as, purified into, and/orused as a trimer. In some embodiments, an IL4/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as a tetramer. Insome embodiments, an IL4/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as a multimer. In someembodiments, an IL4/IL13 fusion protein is present as, purified into,and/or used as a monomer and a dimer. In some embodiments, an IL4/IL13fusion protein is present as, purified into, and/or used as a monomer, adimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, an IL10/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as monomer. In some embodiments,an IL10/IL13 fusion protein of the disclosure is present as, purifiedinto, and/or used as dimer. In some embodiments, an IL10/IL13 fusionprotein of the disclosure is present as, purified into, and/or used astrimer. In some embodiments, an IL10/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as tetramer. Insome embodiments, an IL10/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as multimer. In some embodiments,an 10/IL13 fusion protein is present as, purified into, and/or used asmonomer and a dimer. In some embodiments, an IL10/IL13 fusion protein ispresent as, purified into, and/or used as monomer, a dimer, a trimer, atetramer, a multimer, or any combination thereof.

In some embodiments, an IL13/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as monomer. In some embodiments,an IL13/IL13 fusion protein of the disclosure is present as, purifiedinto, and/or used as dimer. In some embodiments, an IL13/IL13 fusionprotein of the disclosure is present as, purified into, and/or used astrimer. In some embodiments, an IL13/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as tetramer. Insome embodiments, an IL13/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as multimer. In some embodiments,an IL10/IL13 fusion protein is present as, purified into, and/or used asmonomer and a dimer. In some embodiments, an IL10/IL13 fusion protein ispresent as, purified into, and/or used as monomer, a dimer, a trimer, atetramer, a multimer, or any combination thereof.

In some embodiments, an IL27/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as monomer. In some embodiments,an IL27/IL13 fusion protein of the disclosure is present as, purifiedinto, and/or used as dimer. In some embodiments, an IL27/IL13 fusionprotein of the disclosure is present as, purified into, and/or used astrimer. In some embodiments, an IL27/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as tetramer. Insome embodiments, an IL27/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as multimer. In some embodiments,an IL27/IL13 fusion protein is present as, purified into, and/or used asmonomer and a dimer. In some embodiments, an IL27/IL13 fusion protein ispresent as, purified into, and/or used as monomer, a dimer, a trimer, atetramer, a multimer, or any combination thereof.

In some embodiments, an IL33/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as monomer. In some embodiments,an IL33/IL13 fusion protein of the disclosure is present as, purifiedinto, and/or used as dimer. In some embodiments, an IL33/IL13 fusionprotein of the disclosure is present as, purified into, and/or used astrimer. In some embodiments, an IL33/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as tetramer. Insome embodiments, an IL33/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as multimer. In some embodiments,an IL33/IL13 fusion protein is present as, purified into, and/or used asmonomer and a dimer. In some embodiments, an IL33/IL13 fusion protein ispresent as, purified into, and/or used as monomer, a dimer, a trimer, atetramer, a multimer, or any combination thereof.

In some embodiments, an TGF61/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as monomer. In some embodiments,a TGF61/IL13 fusion protein of the disclosure is present as, purifiedinto, and/or used as dimer. In some embodiments, a TGFβ1/IL13 fusionprotein of the disclosure is present as, purified into, and/or used astrimer. In some embodiments, a TGF61/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as tetramer. Insome embodiments, a TGFβ1/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as multimer. In some embodiments,a TGF61/IL13 fusion protein is present as, purified into, and/or used asmonomer and a dimer. In some embodiments a TGFβ1/IL13 fusion protein ispresent as, purified into, and/or used as monomer, a dimer, a trimer, atetramer, a multimer, or any combination thereof.

In some embodiments, a TGFβ2/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as monomer. In some embodiments,a TGFβ2/IL13 fusion protein of the disclosure is present as, purifiedinto, and/or used as dimer. In some embodiments, a TGFβ2/IL13 fusionprotein of the disclosure is present as, purified into, and/or used astrimer. In some embodiments, a TGFβ2/IL13 fusion protein of thedisclosure is present as, purified into, and/or used as tetramer. Insome embodiments, an TGFβ2/IL13 fusion protein of the disclosure ispresent as, purified into, and/or used as multimer. In some embodiments,a TGFβ2/IL13 fusion protein is present as, purified into, and/or used asmonomer and a dimer. In some embodiments, a TGFβ2/IL13 fusion protein ispresent as, purified into, and/or used as monomer, a dimer, a trimer, atetramer, a multimer, or any combination thereof.

Methods of Making

The fusion protein of the present invention may be prepared bytechniques which are routine to the skilled person. For example, it maybe prepared using a technique which provides for the production ofrecombinant fusion proteins by continuous cell lines in culture. Forexample, fusion proteins of the present invention can be produced in ahost cell transfectoma using a combination of recombinant DNA techniquesand gene transfection methods.

For example, to express the fusion proteins of the present invention, anucleic acid molecule encoding the fusion proteins of the presentinvention can be prepared by standard molecular biology techniques. Thenucleic acid molecule of the invention is preferably operably linked totranscription regulatory sequences such as a promoter, and optionally a3′ untranslated region. The nucleic acid molecule of the presentinvention may be inserted into a vector, such as an expression vector,such that the genes are operatively linked to transcriptional andtranslational control sequences. The expression vector and transcriptionregulatory sequences are selected to be compatible with the expressionhost cell used. The nucleic acid molecule encoding a fusion protein ofthe present invention may be inserted into the expression vector byroutine methods. The nucleic acid molecule or vector of the presentinvention may further include a nucleotide sequence encoding a signalpeptide, which may facilitate secretion of the fusion protein from thehost cell. Said nucleotide sequence encoding a signal peptide may beoperably linked to the nucleic acid molecule of the present invention.Preferably, said signal peptide is located at the amino terminus of thefusion protein of the present invention, and as such, the nucleotidesequence encoding said signal peptide may be located 5′ of the nucleicacid molecule encoding the fusion protein of the present invention. Thesignal peptide may be a cytokine signal peptide or a signal peptide froma non-cytokine protein. The signal peptide can be absent on a matureform of fusion protein. The promoter may be constitutive or inducible.The vector may comprise a selectable marker for selection of avector-carrying host cell. The vector may comprise an origin ofreplication when the vector is a replicable vector.

The fusion protein according to the invention may be synthesized de novoby chemical synthesis (using e.g. a peptide synthesizer such as suppliedby Applied Biosystems) or may be produced by recombinant host cells byexpressing the nucleic acid sequence encoding the fusion protein,fragment derivative, or variant. Variants and fragments are preferablyfunctional, i.e., they bind at least to one, two, three, four, or all ofthe corresponding membrane-receptor(s) or receptor subunits and have IL4(or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and/or IL13 activity,preferably IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13activity.

The functional activity of IL4 (or IL10 or IL27 or IL33 or TGFβ1 orTGFβ2) and IL13, as well as the IL4 (or IL10 or IL27 or IL33 or TGFβ1 orTGFβ2) and IL13 comprising fusion protein can be determined usingroutine methods known for those skilled in the art. For example, asuitable assay for functionality of IL4, as well as the IL4 (or IL10)and IL13 comprising fusion protein, is the lipopolysaccharide (LPS)induced cytokine release (IL1, IL6, IL8, TNFα) in whole blood,optionally in the presence of anti-IL10 antibody. Functional activitymay also be determined by assessing the activation of intracellularsignalling pathways upon incubation of target cells with the fusionprotein in presence or absence of blocking antibody against eithercytokine moiety of the fusion protein or their receptors.

In another aspect, isolated nucleic acid sequences encoding any of theabove fusion proteins are provided, such as cDNA, genomic DNA and RNAsequences. Due to the degeneracy of the genetic code various nucleicacid sequences may encode the same amino acid sequence. Any nucleic acidsequence encoding the fusion proteins of the invention are hereinreferred to as “IL4/IL13 (or IL10/IL13, IL27/IL13, IL33/IL13,TGFβ1/IL13, TGFβ2/IL13, IL13/IL13) encoding nucleic acid sequences”. Thenucleic acid sequences provided include recombinant, artificial orsynthetic nucleic acid sequences. It is understood that when sequencesare depicted as DNA sequences while RNA is referred to, the actual basesequence of the RNA molecule is identical with the difference thatthymine (T) is replaced by uracil (U). The nucleic acid sequences of theinvention are particularly useful for expression of the IL4/IL13 (orIL10/IL13, IL27/IL13, IL33/IL13, TGFβ1/IL13, TGFβ2/IL13, IL13/IL13)fusion protein of the invention, for either the production of theseproteins, or for gene therapy purposes.

The nucleic acid sequence, particularly DNA sequence, encoding theIL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/13 or TGFβ1/IL13 orTGFβ2/IL13 or IL13/IL13) fusion protein of this invention can beinserted in expression vectors to produce (e.g., high amounts of)IL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/13 or TGFβ1/IL13 orTGFβ2/IL13, or IL13/IL13) fusion protein. Suitable vectors include,without limitation, linear nucleic acids, plasmids, phagemids, cosmids,RNA vectors, viral vectors and the like. Non-limiting examples of aviral vector include a retrovirus, an adenovirus, and anadeno-associated virus.

Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter.

In addition to the nucleic acid molecules encoding IL4/IL13 (orIL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/113 or TGFβ2/IL13 orIL13/IL13) fusion proteins and regulatory sequences, the recombinantexpression vectors of the invention may carry additional sequences, suchas sequences that regulate replication of the vector in host cells(e.g., origins of replication) and selectable marker genes. Theselectable marker gene facilitates selection of host cells into whichthe vector has been introduced (see e.g., U.S. Pat. No. 4,399,216, U.S.Pat. No. 4,634,665 and U.S. Pat. No. 5,179,017, all by Axel et al.). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection). Finally, the recombinant expression vector may containa gene that codes for a glycosyl transferase in addition to the nucleicacid sequence encoding the fusion proteins of the present invention.

In another aspect, the present invention relates to a host cellcomprising a nucleic acid sequence of the present invention, or anucleic acid construct or vector comprising a nucleic acid sequence ofthe present invention. The host cell may be any host cell. The host cellmay be selected from prokaryotic and eukaryotic cells. The host cell mayalso be a cell line, such as a prokaryotic or eukaryotic cell line. Thehost cell is preferably an animal cell or cell line, such as a mammaliancell or cell line.

In one embodiment the fusion proteins of the present invention areexpressed in eukaryotic cells, such as mammalian host cells. Preferredmammalian host cells for expressing the fusion proteins of the inventioninclude CHO cells (including dhfr-CHO cells, described in (Urlaub etal., 1980), used with a DHFR selectable marker, NS/0 myeloma cells, COScells, HEK293 cells, PER.C6 cells, SP2.0 cells, or other cells. Whenrecombinant expression vectors comprising nucleic acid sequencesencoding a fusion protein according to the present invention areintroduced into mammalian host cells, the fusion proteins of the presentinvention may be produced by culturing the host cells for a period oftime sufficient to allow for expression of the fusion proteins in thehost cells or, more preferably, secretion of the fusion proteins intothe culture medium in which the host cells are grown. The fusionproteins of the present invention can be recovered from the culturemedium using standard protein purification methods.

Alternatively, the nucleic acid sequences encoding the fusion proteinsof the invention can be expressed in other expression systems, includingprokaryotic cells, such as microorganisms, e.g. E. coli, or in algalexpression systems, insect cell expression systems, or cell-free proteinsynthesis systems. Furthermore, the fusion proteins of the presentinvention can be produced in transgenic non-human animals, such as inmilk from sheep and rabbits or eggs from hens, or in transgenic plants.

Introduction of the nucleic acid sequence of the present invention intoa host cell may be carried out by any standard technique known in theart. For expression of the fusion proteins of the present invention, theexpression vector(s) encoding the fusion protein may transfected into ahost cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection, lipofectamine transfection andfreeze-dry method transfection, and the like. Cell lines that secretefusion proteins of the present invention can be identified by assayingculture supernatants for the presence of the fusion protein. Thepreferred screening procedure comprises two sequential steps, the firstbeing identification of cell lines that secrete the fusion protein, thesecond being determination of the quality of the fusion protein such asthe ability of the fusion protein to inhibit cytokine production byblood cells stimulated with LPS or other Toll-like receptor agonists,glycosylation patterns, and other.

For optimal expression in a host cell, the DNA sequence encoding afusion protein of the disclosure (e.g., an IL4/IL13 or IL10/IL13 orIL27/IL13 or IL33/13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13 fusionprotein can be codon-optimized by adapting the codon usage to that mostpreferred in host cell genes. Several techniques for modifying the codonusage to that preferred by the host cells can be found in patent andscientific literature. The exact method of codon usage modification isnot critical for this invention.

In another embodiment of the invention PCR primers and/or probes andkits for detecting the IL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/IL13or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion protein encoding DNA orRNA sequences are provided. Degenerate or specific PCR primer pairs toamplify IL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13or TGFβ2/IL13 or IL13/IL13) fusion protein encoding DNA from samples canbe synthesized (see Dieffenbach and Dveksler (1995) PCR Primer: ALaboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson atal. (2000) PCR-Basics: From Background to Bench, First Edition, SpringerVerlag, Germany). For example, any stretch of 9, 10, 11, 12, 13, 14, 15,16, 18 or more contiguous nucleotides of an IL4/IL13 (or IL10/IL13 orIL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusionprotein encoding nucleic acid sequence (or the complement strand) may beused as primer or probe. Likewise, DNA fragments of an IL4/IL13 (orIL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 orIL13/IL13) fusion protein encoding nucleic acid sequence can be used ashybridization probes. A detection kit for an IL4/IL13 (or IL10/IL13 orIL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusionprotein encoding nucleic acid sequence may comprise primers specific foran IL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13 orTGFβ2/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequenceand/or probes specific for an IL4/IL13 (or IL10/IL13 or IL27/IL13 orIL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion proteinencoding nucleic acid sequences, and an associated protocol to use theprimers or probe to detect specifically IL4/IL13 (or IL10/IL13 orIL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusionprotein encoding nucleic acid sequence in a sample. Such a detection kitmay, for example, be used to determine, whether a host cell has beentransformed with a specific an IL4/IL13 (or IL10/IL13 or IL27/IL13 orIL33/IL13 or TGFβ1/IL13 or TGFβ2/113 or IL13/IL13) fusion proteinencoding nucleic acid sequence of the invention. Because of thedegeneracy of the genetic code, some amino acid codons can be replacedby others without changing the amino acid sequence of the protein.

In an aspect, the present invention is concerned with a method forproducing a fusion protein of the disclosure (e.g., an IL4/IL13 orIL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 orIL13/IL13 fusion protein, said method comprising the steps of culturinga host cell of the present invention under conditions permitting theproduction of the fusion protein (e.g., IL4/IL13 or IL10/IL13 orIL27/IL13 or IL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13 fusionprotein; and optionally, recovering the fusion protein. The skilledperson will be capable of routinely selecting conditions permittingproduction of the IL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/IL13 orTGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion protein. Additionally, aperson skilled in the art will be capable of recovering the fusionprotein produced using routine methods, which include, withoutlimitation, chromatographic methods (including, without limitation, sizeexclusion chromatography, hydrophobic interaction chromatography, ionexchange chromatography, affinity chromatography, immunoaffinitychromatography, metal binding, and the like), immunoprecipitation, HPLC,ultracentrifugation, precipitation and differential solubilisation, andextraction. As said above, recovery or purification of the fusionprotein may be facilitated by adding, for example, a His-tag to thefusion protein.

Pharmaceutical Composition

In an aspect, the invention relates to a pharmaceutical compositioncomprising the fusion protein of the present invention and apharmaceutically acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques (e.g., asdescribed in Remington: The Science and Practice of Pharmacy, 19^(th)Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995).

The term “pharmaceutically acceptable carrier” relates to carriers orexcipients, which are inherently nontoxic and nontherapeutic. Examplesof such excipients are, but are not limited to, saline, Ringer'ssolution, dextrose solution and Hank's solution.

The pharmaceutical composition may be administered by any suitable routeand mode. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults.

The pharmaceutical compositions according to the invention may beformulated in accordance with routine procedures for administration byany route, preferably parenteral. The compositions may be in the form ofliquid preparations, such as oral or sterile parenteral solutions orsuspensions.

The pharmaceutical compositions of the present invention include thosesuitable for any form of parenteral administration.

In an embodiment, the pharmaceutical composition is administeredparenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural intrasternal, intracerebral,intraocular, intralesional, intracerebroventricular, intracisternal, andintraparenchymal, e.g., injection and infusion.

In an embodiment the pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

In an embodiment the fusion proteins of the invention are administeredin crystalline form by subcutaneous injection.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical composition ofthe invention is contemplated. Preferably, the carrier is suitable forparenteral administration, e.g. intravenous or subcutaneous injection orinfusion.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. Examples of suitableaqueous and non-aqueous carriers which may be employed in thepharmaceutical compositions of the invention include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients e.g. as enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients e.g. from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Depending on the route of administration, the active compound, i.e., theIL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/IL13 or TGFβ1/IL13 orTGFβ2/IL13 or IL13/IL13) fusion protein, may be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate the compound. For example, the compound may beadministered to a subject in an appropriate carrier, for example,liposomes. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., 1984).

The fusion proteins of the present invention may also be prepared withcarriers that will protect it against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for the preparation of such formulations are generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

A pharmaceutical composition of the disclosure can be a combination ofany pharmaceutical compounds described herein with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism.

Pharmaceutical formulations for administration can include aqueoussolutions of the active compounds in water soluble form. Suspensions ofthe active compounds can be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension canalso contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. The active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the compounds described herein areadministered in pharmaceutical compositions to a subject having adisease or condition to be treated. In some embodiments, the subject isa mammal such as a human. A therapeutically-effective amount can varywidely depending on the severity of the disease, the age and relativehealth of the subject, the potency of the compounds used, and otherfactors.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising compounds described herein can bemanufactured, for example, by mixing, dissolving, emulsifying,encapsulating, entrapping, or compression processes.

The pharmaceutical compositions can include at least onepharmaceutically-acceptable carrier, diluent, or excipient and compoundsdescribed herein as free-base or pharmaceutically-acceptable salt form.Pharmaceutical compositions can contain solubilizers, stabilizers,tonicity enhancing agents, buffers, and preservatives.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically-acceptable excipients or carriers to form asolid, semi-solid, or liquid composition. Solid compositions include,for example, powders, dispersible granules, and cachets. Liquidcompositions include, for example, solutions in which a compound isdissolved, emulsions comprising a compound, or a solution containingliposomes, micelles, or nanoparticles comprising a compound as disclosedherein. Semi-solid compositions include, for example, gels, suspensionsand creams. The compositions can be in liquid solutions or suspensions,solid forms suitable for solution or suspension in a liquid prior touse, or as emulsions. These compositions can also contain minor amountsof nontoxic, auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, and other pharmaceutically-acceptableadditives.

Non-limiting examples of dosage forms suitable for use in the disclosureinclude liquid, powder, gel, nanosuspension, nanoparticle, microgel,aqueous or oily suspensions, emulsion, and any combination thereof.

Non-limiting examples of pharmaceutically-acceptable excipients suitablefor use in the disclosure include binding agents, disintegrating agents,anti-adherents, anti-static agents, surfactants, anti-oxidants, coatingagents, coloring agents, plasticizers, preservatives, suspending agents,emulsifying agents, anti-microbial agents, spheronization agents, andany combination thereof.

Non-limiting examples of pharmaceutically-acceptable carriers includesaline, Ringer's solution, and dextrose solution. In some embodiments,the pH of the solution can be from about 5 to about 8, and can be fromabout 7 to about 7.5. Further carriers include sustained releasepreparations such as semipermeable matrices of solid hydrophobicpolymers containing the compound. The matrices can be in the form ofshaped articles, for example, films, liposomes, microparticles, ormicrocapsules.

The pH of the disclosed composition can range from about 3 to about 12.The pH of the composition can be, for example, from about 3 to about 4,from about 4 to about 5, from about 5 to about 6, from about 6 to about7, from about 7 to about 8, from about 8 to about 9, from about 9 toabout 10, from about 10 to about 11, or from about 11 to about 12 pHunits. The pH of the composition can be, for example, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, orabout 12 pH units. The pH of the composition can be, for example, atleast 3, at least 4, at least 5, at least 6, at least 6.2 at least 6.4,at least 6.6, at least 6.8, at least 7, at least 7.2, at least 7.4, atleast 7.6, at least 7.8, at least 8, at least 9, at least 10, at least11 or at least 12 pH units. The pH of the composition can be, forexample, at most 3, at most 4, at most 5, at most 6, at most 6.2 at most6.4, at most 6.6, at most 6.8, at most 7, at most 7.2, at most 7.4, atmost 7.6, at most 7.8, at most 8, at most 9, at most 10, at most 11, orat most 12 pH units. A pharmaceutical formulation disclosed herein canhave a pH of from about 5.5 to about 8.5.

Formulations of the disclosure can comprise sugars, alcohols,antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. These compositions can also containpreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms upon the subject compoundscan be achieved by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It can also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin. If desired, the formulation can bediluted prior to use with, for example, an isotonic saline solution or adextrose solution.

In some embodiments, the pharmaceutical composition provided hereincomprises a therapeutically effective amount of a compound (e.g., fusionprotein) in admixture with a pharmaceutically-acceptable carrier and/orexcipient, for example, saline, phosphate buffered saline, phosphate andamino acids, polymers, polyols, sugar, buffers, preservatives, and otherproteins. Illustrative agents include octylphenoxy polyethoxy ethanolcompounds, polyethylene glycol monostearate compounds, polyoxyethylenesorbitan fatty acid esters, sucrose, fructose, dextrose, maltose,glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol,lactose, trehalose, bovine or human serum albumin, citrate, acetate,Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine,glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene,and glycol.

In some embodiments, a pharmaceutical formulation disclosed herein cancomprise: (i) a compound or fusion protein disclosed herein; (ii) abuffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and (v) astabilizer. In some embodiments, the pharmaceutical formulationdisclosed herein is a stable liquid pharmaceutical formulation.

For solid compositions, solid carriers include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talc, cellulose, glucose, sucrose, and magnesiumcarbonate.

A pharmaceutical carrier or excipient can be a solvent, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like that is physiologicallycompatible. The carrier can be suitable for administration by a routedisclosed herein (e.g., parenteral).

A composition of the invention can be, for example, an immediate releaseform or a controlled release formulation. An immediate releaseformulation can be formulated to allow the compounds to act rapidly.Non-limiting examples of immediate release formulations include readilydissolvable formulations. A controlled release formulation can be apharmaceutical formulation that has been adapted such that release ratesand release profiles of the active agent can be matched to physiologicaland chronotherapeutic requirements or, alternatively, has beenformulated to effect release of an active agent at a programmed rate.Non-limiting examples of controlled release formulations includegranules, delayed release granules, hydrogels (e.g., of synthetic ornatural origin), other gelling agents (e.g., gel-forming dietaryfibers), matrix-based formulations (e.g., formulations comprising apolymeric material having at least one active ingredient dispersedthrough), granules within a matrix, polymeric mixtures, and granularmasses.

In some embodiments, a formulation of the disclosure contains a thermalstabilizer, such as a sugar or sugar alcohol, for example, sucrose,sorbitol, glycerol, trehalose, or mannitol, or any combination thereof.In some embodiments, the stabilizer is a sugar. In some embodiments, thesugar is sucrose, mannitol or trehalose.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins1999), each of which is incorporated by reference in itsentirety.

A pharmaceutical composition can be administered in a local manner, forexample, via injection of the compound directly into an organ,optionally in a depot or sustained release formulation or implant. Apharmaceutical composition can be provided in the form of a rapidrelease formulation, in the form of an extended release formulation, orin the form of an intermediate release formulation. A rapid release formcan provide an immediate release. An extended release formulation canprovide a controlled release or a sustained delayed release.

In some embodiments, a pump can be used for delivery of thepharmaceutical composition. In some embodiments, a pen delivery devicecan be used, for example, for subcutaneous delivery of a composition ofthe disclosure. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device can use a replaceablecartridge that contains a pharmaceutical composition disclosed herein.Once all of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.A disposable pen has no replaceable cartridge. Rather, the disposablepen delivery device comes prefilled with the pharmaceutical compositionheld in a reservoir within the device. Once the reservoir is emptied ofthe pharmaceutical composition, the entire device is discarded.

A pharmaceutical composition described herein can be in a unit dosageform suitable for a single administration of a precise dosage. In unitdosage form, the formulation can be divided into unit doses containingappropriate quantities of one or more compounds, fusion proteins, and/ortherapeutic agents. The unit dosage can be in the form of a packagecontaining discrete quantities of the formulation. Non-limiting examplesare packaged injectables, vials, and ampoules. An aqueous suspensioncomposition disclosed herein can be packaged in a single-dosenon-reclosable container. Multiple-dose reclosable containers can beused, for example, in combination with or without a preservative. Aformulation for injection disclosed herein can be present in a unitdosage form, for example, in ampoules, or in multi dose containers witha preservative.

In some embodiments, a pharmaceutical formulation disclosed herein is aliquid formulation that can comprise about 50 μg/mL to about 100 mg/mLof fusion protein. A formulation can comprise, for example, at least 50μg/mL, at least 100 μg/mL, at least 200 μg/mL, at least 300 μg/mL, atleast 400 μg/mL, at least 500 μg/mL, at least 600 μg/mL, at least 700μg/mL, at least 800 μg/mL, at least 900 μg/mL, at least 1 mg/mL, atleast 10 mg/mL, at least 20 mg/mL, or at least 50 mg/mL. In someembodiments, a formulation can comprise at most 100 μg/mL, at most 200μg/mL, at most 300 μg/mL, at most 400 μg/mL, at most 500 μg/mL, at most600 μg/mL, at most 700 μg/mL, at most 800 μg/mL, at most 900 μg/mL, atmost 1 mg/mL, at most 10 mg/mL, at most 20 mg/mL, or at most 50 mg/mL.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Actual dosage levels of the IL4/IL13 (or IL10/IL13 or IL27/IL13 orIL33/IL13 TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion proteins in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the IL4/IL13 (or IL10/IL13 or IL27/IL13 orIL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion protein whichis effective (“effective amount”) to achieve the desired therapeuticresponse for a particular subject (e.g., patient), composition, and modeof administration, without being toxic to the subject (e.g., patient).The selected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

In one embodiment the IL4/IL13 (or IL10/IL13 or IL27/IL13 or IL33/IL13or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion proteins presentinvention can be given as intravenous injection or a short infusion, inanother embodiment, they are administered by slow continuous infusionover a long period, such as more than 24 hours, in order to reduce toxicside effects.

In yet another embodiment, the IL4/IL13 (or IL10/IL13 or IL27/IL13 orIL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion proteins ofthe present invention can be administered as maintenance therapy, suchas, e.g., once a week for a period of 6 months or more.

In another embodiment, the IL4/IL13 (or IL10/IL13 or IL27/IL13 orIL33/IL13 or TGFβ1/IL13 or TGFβ2/IL13 or IL13/IL13) fusion proteins ofthe present invention can be administered as an intrathecal injection.And in yet another embodiment they are administered through anintrathecal or intraspinal drug delivery system which allows continuousor repeated administration.

Therapeutic agents described herein can be administered before, during,or after the occurrence of a disease or condition, and the timing ofadministering the composition containing a therapeutic agent can vary.For example, the compositions can be used as a prophylactic and can beadministered continuously to subjects with a propensity to conditions ordiseases in order to lessen a likelihood of the occurrence of thedisease or condition. The compositions can be administered to a subjectduring or as soon as possible after the onset of the symptoms. Theinitial administration can be via any route practical, such as by anyroute described herein using any formulation described herein. Atherapeutic agent can be administered as soon as is practicable afterthe onset of a disease or condition is detected or suspected, and for alength of time necessary for the treatment of the disease, such as, forexample, from about 1 month to about 3 months. The length of treatmentcan vary for each subject.

A dose can be based on the amount of the fusion protein per kilogram ofbody weight of a subject. A dose a of a fusion protein can be at leastabout 0.5 μg/kg, 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 p μg/kg, 125μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg,300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg,625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg,950 μg/kg, 975 μg/kg, or 1 mg/kg. A dose a of a fusion protein can be atmost about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 p μg/kg, 125μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg,300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg,625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg,950 μg/kg, 975 μg/kg, or 1 mg/kg.

In some embodiments, a dose can be at least about 1 ng, at least 10 ng,at least 100 ng, at least 500 ng, at least at least 1 μg, at least 5 μg,at least at least 10 μg, at least 50 μg, at least at least 100 μg, atleast 500 μg, at least at least 1 mg, at least 5 mg, at least 10 mg, atleast 50 mg, or at least 100 mg. A dose can be at most about 1 ng, atmost 10 ng, at most 100 ng, at most 500 ng, at most at most 1 μg, atmost 5 μg, at most at most 10 μg, at most 50 μg, at most at most 100 μg,at most 500 μg, at most at most 1 mg, at most 5 mg, at most 10 mg, atmost 50 mg, or at most 100 mg.

A dose can be determined by reference to a plasma concentration or alocal concentration of the fusion protein. A target plasma concentrationor local concentration of the fusion protein can be at least about 1 pM,at least about 10 pM, at least about 20 pM, at least about 30 pM, atleast about 40 pM, at least about 50 pM, at least about 60 pM, at leastabout 70 pM, at least about 80 pM, at least about 90 pM, at least about100 pM, at least about 200 pM, at least about 300 pM, at least about 400pM, at least about 500 pM, at least about 600 pM, at least about 700 pM,at least about 800 pM, at least about 900 pM, at least about 1 nM, atleast about 2 nM, at least about 3 nM, at least about 4 nM, at leastabout 5 nM, at least about 6 nM, at least about 7 nM, at least about 8nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, atleast about 30 nM, at least about 40 nM, at least about 50 nM, at leastabout 60 nM, at least about 70 nM, at least about 80 nM, at least about90 nM, at least about 100 nM, at least about 200 nM, at least about 300nM, at least about 400 nM, at least about 500 nM, at least about 600 nM,at least about 700 nM, at least about 800 nM, at least about 900 nM, atleast about 1 μM, at least about 10 μM, or at least about 100 μM. Atarget plasma concentration or local concentration of the fusion proteincan be at most about 1 nM, at most about 10 nM, at most about 100 nM, atmost about 1 μM, at most about 10 μM, at most about 100 μM, or at mostabout 1 mM.

Administration of the fusion protein can continue for as long asclinically necessary. In some embodiments, a fusion protein of thedisclosure can be administered for more than 1 day, more than 1 week,more than 1 month, more than 2 months, more than 3 months, more than 4months, more than 5 months, more than 6 months, more than 7 months, morethan 8 months, more than 9 months, more than 10 months, more than 11months, more than 12 months, more than 13 months, more than 14 months,more than 15 months, more than 16 months, more than 17 months, more than18 months, more than 19 months, more than 20 months, more than 21months, more than 22 months, more than 23 months, or more than 24months. In some embodiments, a fusion protein of the disclosure isadministered for less than 1 week, less than 1 month, less than 2months, less than 3 months, less than 4 months, less than 5 months, lessthan 6 months, less than 7 months, less than 8 months, less than 9months, less than 10 months, less than 11 months, less than 12 months,less than 13 months, less than 14 months, less than 15 months, less than16 months, less than 17 months, less than 18 months, less than 19months, less than 20 months, less than 21 months, less than 22 months,less than 23 months, or less than 24 months.

In some embodiments, a fusion protein can be administered to a subject1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20times over a treatment cycle. In some embodiments, a treatment cycle is7 days, 14 days, 21 days, or 28 days long. In some embodiments, atreatment cycle is 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months,13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 11 months, 22 months, 23 months, 24 months, 25months, 26 months, 27 months, 28 months, 29 months, 30 months, 31months, 32 months, 33 months, 34 months, 35 months, 36 months, 37months, 38 months, 39 months, 40 months, 41 months, 42 months, 43months, 44 months, 45 months, 46 months, 47 months, 48 months, 49months, 50 months, 51 months, 52 months, 53 months, 54 months, 55months, 56 months, 57 months, 58 months, 59 months, or 60 months.

In some embodiments, a fusion protein is administered to a subject onceevery 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

Therapeutic Uses

In a further aspect, the present invention relates to the fusion proteinof the present invention or a nucleic acid encoding the same for use asa medicament.

In an aspect, the present invention pertains to a fusion protein or anucleic acid encoding the same for use in treating pain, for example,chronic pain. “Chronic” may mean that the pains persists/persisted overmore than 2 weeks or more than 1, 3, 6, 12 months, or even more than 1,2, 4, 6 years. Pain can include pain that is mediated by the centralnervous system, the peripheral nervous system, or a combination thereof.Non-limiting types of pain include nociceptive pain, peripheralneuropathic pain, central neuropathic pain, and mixed types of pain.

Neuropathy can contribute to pain. In some embodiments, a fusion proteinof the disclosure can be used for treating a neuropathy. A neuropathycan be associated with pain, numbness, weakness, or a combinationthereof.

Particularly, it was found that the fusion protein of the presentinvention has a long-lasting analgesic effect against allodyniaassociated with chemotherapy-associated neuropathy and neurodegenerationin mice. In addition, the fusion protein prevents neurodegenerationinduced by chemotherapy in vitro and in vivo. Therefore, the fusionprotein of the invention or a nucleic acid encoding the same may be usedfor prevention and treatment neuropathic pain. As such, the fusionprotein may be particularly useful for the treatment of neuropathy(e.g., chemotherapy induced neuropathy), and other forms of peripheralneuropathic pain such as post-herpetic neuralgia, trigeminal neuralgia,post-traumatic or post-operative peripheral neuropathy, diabeticperipheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, painful peripheral neuropathy, nerveentrapment syndrome, chemotherapy-associated pain, complex regional painsyndrome, and other. In some embodiments, a fusion protein of thedisclosure can be used to treat postoperative cognitive dysfunction.

In some embodiments, a fusion protein of the disclosure can be used totreat a condition associated with cancer or chemotherapy, for example,chemotherapy-induced neuropathy, chemotherapy-associated pain, chemobrain, cancer-related cognitive impairment, cancer-related cognitivedysfunction. In some embodiments, the condition is associated with achemotherapy that is being used to treat acute leukemia, astrocytomas,biliary cancer (cholangiocarcinoma), bone cancer, breast cancer, brainstem glioma, bronchioloalveolar cell lung cancer, cancer of the adrenalgland, cancer of the anal region, cancer of the bladder, cancer of theendocrine system, cancer of the esophagus, cancer of the head or neck,cancer of the kidney, cancer of the parathyroid gland, cancer of thepenis, cancer of the pleural/peritoneal membranes, cancer of thesalivary gland, cancer of the small intestine, cancer of the thyroidgland, cancer of the ureter, cancer of the urethra, carcinoma of thecervix, carcinoma of the endometrium, carcinoma of the fallopian tubes,carcinoma of the renal pelvis, carcinoma of the vagina, carcinoma of thevulva, cervical cancer, chronic leukemia, colon cancer, colorectalcancer, cutaneous melanoma, ependymoma , epidermoid tumors, Ewingssarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, glioma,hematologic malignancies, hepatocellular (liver) carcinoma, hepatoma,Hodgkin's Disease, intraocular melanoma, Kaposi sarcoma, lung cancer,lymphomas, medulloblastoma, melanoma, meningioma, mesothelioma, multiplemyeloma, muscle cancer, neoplasms of the central nervous system (CNS),neuronal cancer, small cell lung cancer, non-small cell lung cancer,osteosarcoma, ovarian cancer, pancreatic cancer, pediatric malignancies,pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma,sarcoma of soft tissue, schwanoma, skin cancer, spinal axis tumors,squamous cell carcinomas, stomach cancer, synovial sarcoma, testicularcancer, uterine cancer, or tumors and their metastases, includingrefractory versions of any of the above cancers, or any combinationthereof.

In a further embodiment, the fusion protein of the present invention ora nucleic acid encoding the same may be used to treat pain andneurodegeneration of central neuropathic disorders including spinal cordinjury, post-stroke pain and multiple sclerosis. In some embodiments, afusion protein of the disclosure can be used to treat postoperativecognitive dysfunction.

In a further aspect, the present invention pertains to a fusion proteinof the present invention or a nucleic acid encoding the same for use intreatment of chronic mixed nociceptive and neuropathic pain such as lowback pain, osteoarthritis, cancer pain, chronic visceral pain,fibromyalgia, polymyalgia rheumatica, myofascial pain syndromes, andother.

In yet a further aspect, the fusion protein of the present invention ora nucleic acid encoding the same may be used to treat post-operativeorthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome,inflammatory bowel disease, rheumatoid arthritis, ankylosingspondylitis, and other.

In an embodiment, the condition treated with the fusion protein of thepresent invention is characterized by pain and may be selected fromnociceptive pain, neuropathic pain, or mixed nociceptive-neuropathicpain.

In another embodiment, the fusion protein of the present invention or anucleic acid encoding the same may be used to prevent chronic pain.Particularly, the fusion protein may be used to prevent neuropathic painand neurodegeneration in cancer patients treated with chemotherapy.

In another aspect, the invention is directed to a fusion protein of thepresent invention for use in the prevention or treatment of a clinicalcondition in a mammal, such as a human, for which interleukin 13 isindicated.

In a further aspect, the invention is directed to a fusion protein ofthe present invention for use in the prevention or treatment of aclinical condition in a mammal, such as a human, for which interleukin 4or interleukin 10 or 27 or IL33 or TGFβ1 or TGFβ2 is indicated.

According to one embodiment the fusion proteins taught herein can beused for inhibiting neuro-inflammation due to activation of glial cells,and neuronal cells, infiltrating immune cells, or any combinationthereof in the central nerve system.

As a result, the fusion proteins of the present invention can be usedfor the preparation of a medicament to attenuate neuro-inflammatoryreactions by inhibiting the activation of glial cells and neuronal cellsin vivo.

In an embodiment the fusion proteins of the present invention or nucleicacids encoding the same can be used as stand-alone drug. In anotherembodiment they are used in combination with other drugs. In someembodiments, a fusion protein of the disclosure is administered incombination with an analgesic, for example, acetaminophen/paracetamol, anon-steroidal anti-inflammatory drug (NSAID), or an opioid. In someembodiments, a fusion protein of the disclosure can be used incombination with NSAIDs, such as aspirin, ibuprofen, naproxen,celecoxib, ketorolac, or diclofenac. In some embodiments, a fusionprotein of the disclosure can be used in combination with specific COX-2inhibitors, such as celecoxib (Celebrex®), rofecoxib, or etoricoxib. Insome embodiments, a fusion protein of the disclosure can be used incombination with corticosteroids, such as dexamethasone or glucosteroids(e.g., hydrocortisone and prednisone). In some embodiments, a fusionprotein of the disclosure is administered in combination with anantagonist of a pro-inflammatory cytokine (e.g., and antibodyderivative, or other molecule thereof that binds to TNF-α (e.g.,adalimumab, etanercept), IL-17 (e.g., secukinumab), IL-23 (e.g.,guselkumab, ildrakizumab), or IL-12 and IL-23 (e.g., ustekinumab). Insome embodiments, a fusion protein is administered in combination withhyaluronic acid. Multiple therapeutic agents can be administered in anyorder or simultaneously. In some embodiments, a compound of theinvention is administered in combination with, before, or after anotherdrug.

In some embodiments, a fusion protein of the disclosure or a nucleicacid encoding the same can be used for treating Alpers' Disease,Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, AutoimmuneAtrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular Necrosis,Back Pain, Batten Disease, Behçet's Disease (Syndrome), BreakthroughPain, Burning Mouth Syndrome, Bursitis, Central Autosomal DominantArteriopathy with Subcortical Infarcts and Leukoencephalopathy(Cadasil), Cerebral ischemia, Cerebro-Oculo-Facio-Skeletal Syndrome(COFS), Carpal Tunnel syndrome, Cauda Equina Syndrome, Central PainSyndrome, Cerebral Palsy, Cerebrospinal Fluid (CSF) Leaks, CervicalStenosis, Charcot-Marie-Tooth (CMT) Disease, Chronic FunctionalAbdominal Pain (CFAP), Chronic Pancreatitis, Collapsed Lung(Pneumothorax), Corticobasal Degeneration, Compression injury, CornealNeuropathic Pain, Crush syndrome, Degenerative Disc Disease,Dermatomyositis, Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS),Endometriosis, Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia,Failed Back Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia,Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains,Herniated disc, Hydrocephalus, Intercostal Neuraligia, InterstitialCystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy BodyDementia, Loin Pain-Haematuria Syndrome, Lyme Disease, MeralgiaParesthetica, Mitochondrial Disorders, Mixed dementia, Motor neuronediseases (MND), Monomelic Amyotrophy, Multiple system atrophy (MSA),Myositis, Neck Pain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis,Paget's Disease, Parsonage Turner Syndrome, Pelvic Pain, PeripheralNeuropathy, Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis,Polymyalgia Rhuematica, Polymyositis, Post Herniorraphy Pain Syndrome,Post Mastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy PainSyndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, PsoriaticArthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome,Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis,Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), Herpes Zoster Shingles, SpasmodicTorticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, SpinalStenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, ThoracicOutlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, TraumaticBrain Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combinationthereof.

A fusion protein of the disclosure of a nucleic acid encoding the samecan be used to treat a neuropathy. Non-limiting examples of neuropathyinclude post-traumatic peripheral neuropathy, post-operative peripheralneuropathy, diabetic peripheral neuropathy, inflammatory peripheralneuropathy, HIV-associated neuropathy, chemotherapy-induced neuropathy,polyneuropathy, mononeuropathy, multiple mononeuropathy, cranialneuropathy, predominantly motor neuropathy, predominantly sensoryneuropathy, sensory-motor neuropathy, autonomic neuropathy, idiopathicneuropathy, post-herpetic neuralgia, trigeminal neuralgia,glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia,atypical trigeminal neuralgia, sciatica, brachial plexopathy, orintercostal neuralgia. A neuropathy can be associated with, for example,pain, numbness, weakness, burning, atrophy, tingling, twitching, or acombination thereof.

In some embodiments, a fusion protein of the disclosure or a nucleicacid encoding the same can be used to treat an autoimmune disease.Non-limiting examples of autoimmune diseases include Acute disseminatedencephalomyelitis, Acute motor axonal neuropathy, Addison's disease,Adiposis dolorosa, Adult-onset Still's disease, Alopecia areata,Ankylosing Spondylitis, Anti-Glomerular Basement Membrane nephritis,Anti-neutrophil cytoplasmic antibody-associated vasculitis,Anti-N-Methyl-D-Aspartate Receptor Encephalitis, Antiphospholipidsyndrome, Antisynthetase syndrome, Aplastic anemia, AutoimmuneAngioedema, Autoimmune Encephalitis, Autoimmune enteropathy, Autoimmunehemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease,Autoimmune lymphoproliferative syndrome, Autoimmune neutropenia,Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis,Autoimmune polyendocrine syndrome, Autoimmune polyendocrine syndrometype 2, Autoimmune polyendocrine syndrome type 3, Autoimmuneprogesterone dermatitis, Autoimmune retinopathy, Autoimmunethrombocytopenic purpura, Autoimmune thyroiditis, Autoimmune urticaria,Autoimmune uveitis, Balo concentric sclerosis, Behçet's disease,Bickerstaffs encephalitis, Bullous pemphigoid, Celiac disease, Chronicfatigue syndrome, Chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Coldagglutinin disease, Complex regional pain syndrome, CREST syndrome,Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetesmellitus type 1, Discoid lupus erythematosus, Endometriosis, Enthesitis,Enthesitis-related arthritis, Eosinophilic esophagitis, Eosinophilicfasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essentialmixed cryoglobulinemia, Evans syndrome, Felty syndrome, Fibromyalgia,Gastritis, Gestational pemphigoid, Giant cell arteritis, Goodpasturesyndrome, Graves' disease, Graves ophthalmopathy, Guillain-Barrésyndrome, Hashimoto's Encephalopathy, Hashimoto Thyroiditis,Henoch-Schonlein purpura, Hidradenitis suppurativa, Idiopathic dilatedcardiomyopathy, Idiopathic inflammatory demyelinating diseases, IgAnephropathy, IgG4-related systemic disease, Inclusion body myositis,Inflamatory Bowel Disease (IBD), Intermediate uveitis, Interstitialcystitis, Juvenile Arthritis, Kawasaki's disease, Lambert-Eatonmyasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichensclerosus, Ligneous conjunctivitis, Linear IgA disease, Lupus nephritis,Lupus vasculitis, Lyme disease, Ménière's disease, Microscopic colitis,Microscopic polyangiitis, Mixed connective tissue disease, Mooren'sulcer, Morphea, Mucha-Habermann disease, Multiple sclerosis, Myastheniagravis, Myocarditis, Myositis, Neuromyelitis optica, Neuromyotonia,Opsoclonus myoclonus syndrome, Optic neuritis, Ord's thyroiditis,Palindromic rheumatism, Paraneoplastic cerebellar degeneration, ParryRomberg syndrome, Parsonage-Turner syndrome, Pediatric AutoimmuneNeuropsychiatric Disorder Associated with Streptococcus, Pemphigusvulgaris, Pernicious anemia, Pityriasis lichenoides et varioliformisacuta, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Primary biliary cirrhosis, Primary immunodeficiency, Primarysclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis,Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrenosum,Raynaud's phenomenon, Reactive arthritis, Relapsing polychondritis,Restless leg syndrome, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Rheumatoid vasculitis, Sarcoidosis, Schnitzlersyndrome, Scleroderma, Sjogren's syndrome, Stiff person syndrome,Subacute bacterial endocarditis, Susac's syndrome, Sydenham chorea,Sympathetic ophthalmia, Systemic Lupus Erythematosus, Systemicscleroderma, Thrombocytopenia, Tolosa-Hunt syndrome, Transversemyelitis, Ulcerative colitis, Undifferentiated connective tissuedisease, Urticaria, Urticarial vasculitis, Vasculitis, and Vitiligo.

In some embodiments, a fusion protein of the disclosure or a nucleicacid encoding the same can be used to treat inflammation. In someembodiments, the inflammation is chronic inflammation. In someembodiments, a fusion protein of the disclosure can be used to treatinflammation that is associated with inflammatory bowel disease,irritable bowel syndrome, osteoarthritis, rheumatoid arthritis,glomerulonephritis, sepsis, adult respiratory distress syndrome,dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosingspondylarthritis, systemic lupus erythematosus, vasculitis, gout,allotransplantation, xenotransplantation, an autoimmune disease,Sjogren's disease, a burn injury, trauma, stroke, myocardial infarction,atherosclerosis, diabetes mellitus, extracorporeal dialysis and bloodoxygenation, ischemia-reperfusion injuries, and toxicity induced by thein vivo administration of cytokines or other therapeutic monoclonalantibodies. In some embodiments, the inflammation is chronicinflammation. In some embodiments, a fusion protein of the disclosurecan be used to treat inflammatory bowel disease, irritable bowelsyndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis,sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis,allergic inflammation, psoriasis, ankylosing spondylarthritis, systemiclupus erythematosus, vasculitis, gout, allotransplantation,xenotransplantation, an autoimmune disease, Sjogren's disease, a burninjury, trauma, stroke, myocardial infarction, atherosclerosis, diabetesmellitus, extracorporeal dialysis and blood oxygenation,ischemia-reperfusion injuries, and toxicity induced by the in vivoadministration of cytokines or other therapeutic monoclonal antibodies.

In some embodiments, a fusion protein is used to treat a condition,wherein the condition is not a cancer.

Treatment (prophylactic or therapeutic) will generally consist ofadministering the fusion protein of the present invention or a nucleicacid encoding the same parenterally, preferably intrathecally,intraarticularly, intravenously, intramuscularly or subcutaneously. Thedose and administration regimen will depend on the extent of inhibitionof neuroinflammation and neurodegeneration aimed at. Typically, theamount of the fusion protein given will be in the range of 0.5 μg to 1mg per kg of body weight. The dosage can be determined or adjusted bymeasuring cytokine levels (IL13, IL4 or IL10 or IL27 or IL33 or TGFβ1 orTGFβ2, or a combination thereof) in the body compartment targeted uponadministration. The dose can also be determined by measuringneuro-inflammation in a patient for example by positron emissiontomography (PET) imaging of microglia.

For parenteral administration, the fusion protein or a nucleic acidencoding the same is preferably formulated in an injectable formcombined with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are well-known in the art and examples include saline, dextrosesolution, Ringer's solution and solutions containing small amounts ofhuman serum albumin.

Typically, the fusion proteins of the present invention may beformulated in such vehicles at a concentration of from about 50 μg toabout 100 mg per ml.

Gene Therapy

The nucleic acid constructs or vectors of the present invention may beused as gene therapy agents for treatment of the conditions set forthabove.

As such, in an aspect the invention is directed to a vector as describedabove for use in the prevention or treatment of a conditioncharacterized by visceral or non-visceral nociceptive pain, peripheralor central neuropathic pain, or mixed nociceptive-neuropathic pain,neuroinflammation and/or neurodegeneration, preferably wherein saidcondition may be selected from the group consisting of post-operativeorthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome,inflammatory bowel disease, rheumatoid arthritis, ankylosingspondylitis, post-herpetic neuralgia, trigeminal neuralgia,post-traumatic or post-operative peripheral neuropathy, diabeticperipheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, painful peripheral neuropathy, nerveentrapment syndrome, chemotherapy-associated pain, complex regional painsyndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis,low back pain, osteoarthritis, cancer pain, chronic visceral pain,fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome,Alzheimer's disease and Parkinson's disease, Huntington's disease,and/or amyotrophic lateral sclerosis, or multiple sclerosis. In someembodiments, the condition can be Alpers' Disease, Arachnoiditis,Arthrofibrosis, Ataxic Cerebral Palsy, Autoimmune Atrophic Gastritis,Amyloidosis, hATTR Amyloidosis, Avascular Necrosis, Back Pain, BattenDisease, Behcet's Disease (Syndrome), Breakthrough Pain, Burning MouthSyndrome, Bursitis, Central Autosomal Dominant Arteriopathy withSubcortical Infarcts and Leukoencephalopathy (Cadasil), Cerebralischemia, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnelsyndrome, Cauda Equina Syndrome, Central Pain Syndrome, Cerebral Palsy,Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis, Charcot-Marie-Tooth(CMT) Disease, Chronic Functional Abdominal Pain (CFAP), ChronicPancreatitis, Collapsed Lung (Pneumothorax), Corticobasal Degeneration,Compression injury, Corneal Neuropathic Pain, Crush syndrome,Degenerative Disc Disease, Dermatomyositis, Dementia, Dystonia,Ehlers-Danlos Syndrome (EDS), Endometriosis, Eosinophilia-MyalgiaSyndrome (EMS), Erythromelalgia, Failed Back Surgery Syndrome (FBSS),Fibromyalgia, Friedreich's Ataxia, Frontotemporal dementia,Glossopharyngeal neuralgia, Growing Pains, Herniated disc,Hydrocephalus, Intercostal Neuraligia, Interstitial Cystitis, JuvenileDermatositis, Knee Injury, Leg Pain, Lewy Body Dementia, LoinPain-Haematuria Syndrome, Lyme Disease, Meralgia Paresthetica,Mitochondrial Disorders, Mixed dementia, Motor neurone diseases (MND),Monomelic Amyotrophy, Multiple system atrophy (MSA), Myositis, NeckPain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis, Paget'sDisease, Parsonage Turner Syndrome, Pelvic Pain, Peripheral Neuropathy,Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis, PolymyalgiaRhuematica, Polymyositis, Post Herniorraphy Pain Syndrome, PostMastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy PainSyndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, PsoriaticArthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome,Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis,Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), Herpes Zoster Shingles, SpasmodicTorticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, SpinalStenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, ThoracicOutlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, TraumaticBrain Injuries, Vascular Pain, Vulvodynia, Acute disseminatedencephalomyelitis, Accute Optic Neuritis, Transverse Myelitis,Neuromyelitis Optica, or Whiplash.

As described earlier herein, the present invention relates to a fusionprotein comprising at least 2, 3, 4, preferably 2 regulatory (e.g.,anti-inflammatory) interleukins chosen from the group consisting ofinterleukin 13 (IL13), interleukin 4 (IL4), interleukin 10 (IL10),interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factorbeta 1 (TGFβ1), and transforming growth factor beta 2 (TGFβ2).Accordingly, where reference is made in the present disclosure to“IL13”, this may be replaced by transforming growth factor beta 1(TGFβ1), or transforming growth factor beta 2 (TGFβ2). Similarly, wherereference is made in the present disclosure to “IL4”, this may bereplaced by transforming growth factor beta 1 (TGFβ1), or transforminggrowth factor beta 2 (TGFβ2). The present disclosure thus alsoencompasses a fusion protein of IL13/TGFβ1 or IL13/TGFβ2. Further, thepresent disclosure encompasses a fusion protein of IL4/TGFβ1 orIL4/TGFβ2. Additionally, the present disclosure encompasses a fusionprotein of IL10/TGFβ1 or IL10/TGFβ2.

The present invention will now be illustrated with reference to thefollowing examples, which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES

Animals.

All animal experiments were performed in agreement with internationalguidelines and with prior approval by the University Medical CentreUtrecht experimental animal committee. Experiments were conducted usingboth male and female C57BL/6 mice aged between 8 and 16 weeks. Observerscarrying out behavioural experiments were blinded to treatment.

Material and Methods

Expression and purification of an IL4/IL13 fusion protein. IL4/13 fusionprotein HEK293 cells were transiently transfected according to standardprocedures with a vector containing a transgene (Y Derocher et al.,Nucleic Acids Research 2002, vol 30, no 2, e9). Briefly, synthetic cDNA(GeneArt, ThermoFisher Scientific) coding for an IL4/IL13 fusion proteinsequence of the present invention (see SEQ ID NO: 4, N-terminal of thissequence a 6-His tag was inserted) was cloned in an expression vector,containing a cystatin signal sequence. HEK293E cells were thentransfected with the expression vector, and co-transfected with a vectorcarrying the transgene for beta-galactoside alpha-2,3-sialyltransferase5 (SIAT 9; homo sapiens) to optimize capping of the glycans with sialicacid. Cells were cultured in FreeStyle medium (Invitrogen) with 0.9%primatone and ˜0.04%, v/v, fetal calf serum as described before^(19,26).Cell suspension was collected on day 4 after transfection andcentrifuged at 435×g for 5 minutes. The supernatant was passed through aHIS-Select Nickel Affinity gel (Sigma-Aldrich) to purify the recombinantIL4/IL13 fusion protein. Elution fraction was dialysed overnight at 4°C. against phosphate buffered saline, pH 7.4 (PBS).

Protein assays. Bradford (Bio-Rad), BCA (Thermo Scientific), and Qubit1.0 (Thermo Scientific) were used to determine the amount of protein inthe eluded & dialysed fractions. All protein assays were performedaccording to the manufacturer's protocols.

ELISAs. The amount of fusion protein was determined based on the amountof the individual cytokines, measured with ELISA (IL4 Pelipair ELISAkit, Sanquin; IL13, DuoSet ELISA, R&D Systems). ELISAs were performedaccording to the manufacturer's instructions. Concentrations werecalculated based on the theoretical molecular weight of the fusionprotein compared to the individual cytokines.

SDS-Page and Western Blot. Fractions of the HIS-Select Nickel affinitychromatography purification (load, flow-through, wash, elution/dialysis)were separated on 12% polyacrylamide SDS-Page gels (Bio-Rad) andtransferred to polyvinylidene difluoride membranes. Membranes werestained with IL4 antibody (Santa Cruz, SC-13555).

High Performance Size Exclusion Chromatography (HP-SEC). To determineits molecular weight and homogeneity, IL4/IL13 fusion protein wasanalyzed with High Performance Size Exclusion Chromatography (HP-SEC).The gel filtration (BioSuite 125 4 μm UHR SEC Column; Waters; Cat#186002161) was performed on a High-Performance Liquid ChromatographySystem (Shimadzu) with 50 mM phosphate buffer containing 0.5 M NaCl asmobile phase. The column was calibrated prior to the run using a proteinmix of thyroglobulin, bovine serum albumen, carbonic anhydrase,myoglobulin, and ribonuclease. Fifty μl of 20 μg/ml of purified IL4/IL13fusion protein was injected and separated on the column at a flow rateof 0.35 ml/min and under a pressure of 35 bar.

Evaluation of chemotherapy-induced neurotoxicity in vitro. Dorsal rootganglion (DRG) neurons were cultured as described previously²⁷. Briefly,adult mice DRG neurons were dissected out and subsequently digested inan enzyme mixture containing Ca²⁺- and Mg²⁺-free HBSS, 5 mM HEPES, 10 mMglucose, collagenase type XI (5 mg/ml) and dispase (10 mg/ml) for 1 hourbefore mechanical trituration in DMEM containing 10%, v/v,heat-inactivated fetal calf serum. Cells were centrifuged for 5 min at800 rpm, resuspended in DMEM containing 4.5 g/L glucose, 4 mML-glutamine, 110 mg/L sodium pyruvate, 10% fetal calf serum, 1%penicillin-streptomycin (10,000 IU/ml), 1% glutamax, and incubated in 24wells plates for 24 hours in presence of paclitaxel (1 uM) oroxaliplatin (5 ug/ml) to induce neurotoxicity. IL4/IL10 fusion protein(100 ng/mL), IL4/IL13 fusion protein (100 ng/mL), IL4 an IL13 (50 ng/mLeach), IL4 (50 ng/mL), IL10 (50 ng/mL), and IL13 (50 ng/mL) were addedtogether with the chemotherapeutic agent. As controls cells were alsocultured in absence of chemotherapeutic drugs or cytokines, and inpresence of chemotherapeutic drugs only. After fixation with 4%paraformaldehyde, cells were stained with rabbit anti-mouse βIII-tubulin(ab18207, 1:1000; Abcam). Neurites were visualized with a Zeiss Axio LabAl microscope (Zeiss-Oberkochen, Germany) and using a random samplingmethod, at least 10 images per glass slide were made at a magnificationof 10×. The length of neurites was measured with the ImageJ pluginSimple Neurite Tracer76. The averages of neurite length per neuron for aminimum of five neurons per condition were compared between groups forthe three individual primary sensory cultures.

Chemotherapy-induced polyneuropathy and assessment of allodynia. Toinduce transient chemotherapy-induced polyneuropathy (CIPN), paclitaxel(2 mg/kg, Cayman Chemical Company) was injected intraperitoneally ondays 0 and 2. To induce persistent paclitaxel-induced CIPN, paclitaxel(8 mg/kg, Cayman Chemical Company) was injected intraperitoneal on day0, 2, 4 and 6. To induce persistent oxaliplatin-induced polyneuropathy,mice received two treatment cycles, each consisting of 5 dailyintraperitoneal injections of 3 mg/kg oxaliplatin (Tocris) with a 5 daystreatment-free interval.

Noxious mechanical sensitivity in the hind paws was measured using vonFrey hairs (Stoelting, Wood Dale, USA). Results were expressed as the50% paw-withdrawal threshold using the up-and-down method²⁹. In someexperiments the length of intraepidermal nerve fibers in the paw skin atday 15 was determined by immunofluorescent staining of skin biopsieswith the neuronal marker PGP9.5. All experiments were performed in ablinded manner.

Statistical analysis. Unless indicated otherwise, all data are expressedas mean±SEM. Data were analysed for statistical significance by one-wayor two-way ANOVA (with repeated measures if appropriate) followed by theappropriate post-hoc test. A p value of p<0.05 was consideredsignificant.

Example 1 Endogenous IL4 and IL13 are Necessary for Normal Resolution ofPaclitaxel-Induced Transient Hyperalgesia

To investigate the possible role of regulatory (e.g., anti-inflammatory)cytokines on recovery from chemotherapy-induced polyneuropathy, thetransient pain model of paclitaxel-induced pain¹⁵ was used. Micereceived 2 injections of low dose (2 mg/kg) of paclitaxel on days 0 and2. From days 6 to 10 after start of chemotherapy treatment, micereceived a daily intrathecal injection of neutralizing antibodiesagainst IL4, IL13 or control IgG antibodies (5 μg antibody perinjection; FIG. 1). Mice that received paclitaxel, developed mechanicalhyperalgesia starting on the first day of chemotherapy, which resolvedspontaneously after one week of treatment termination (FIG. 1). Inanimals intrathecally injected with neutralizing antibodies against IL4or IL13, resolution of hyperalgesia was delayed and persisted for atleast 2 weeks. These data indicate that endogenously produced IL4 andIL13 are necessary for normal pain resolution after chemotherapytreatment (FIG. 1).

Example 2 Neuroprotective Effects of IL4, IL10 and IL13

To assess whether regulatory (e.g., anti-inflammatory) cytokines possessneuroprotective properties sensory neurons isolated from the dorsal rootganglion were cultured overnight with paclitaxel in combination ofeither IL4, IL10 or IL13 as described in methods. Neurotoxicity wasevaluated by measuring neurite length using βIII-tubulin staining.Paclitaxel (1 μM) reduced neurite length with ˜50% indicating paclitaxeldamaged sensory neurons. Addition of IL13 during the culture withpaclitaxel prevented the paclitaxel-induced negative effect on neuritelength, whilst IL10 and IL4 did not (FIG. 2).

Example 3 Characterization of Recombinant IL4/13 Fusion Protein

Human IL4/IL13 fusion protein (see SEQ ID NO: 4) with an N-terminal 6His-tag was produced by transient transfection of HEK293 cells andpurified as described in methods. On HP-SEC the purified IL4/IL13 fusionprotein migrated as a single peak with an apparent mass of 40 kDa (FIG.3). The preparation was also analyzed on SDS-PAGE and Coomassiestaining. A homogenous preparation migrating as a smear with a molecularmass of ˜37 kDa was detected (insert FIG. 3).

Example 4 IL4/IL13 Fusion Protein Cures Paclitaxel-Induced PersistentPolyneuropathy

The potential of IL4/IL13 fusion protein to inhibit chemotherapy-inducedhyperalgesia was evaluated in model of persistent paclitaxel-inducedpainful neuropathy³⁰. Mice received 4 injections of paclitaxel (8 mg/kg)every other day from day 0 to 6. Paclitaxel induced mechanicalhyperalgesia that started on the first day after the first injection andthat persisted at least 3 weeks after chemotherapy-treatment wasstopped. Two days after the last paclitaxel injection, mice wereinjected intrathecally with 3 different doses of IL4/IL13 fusion proteincomprising SEQ ID NO: 4 (0.3, 1 and 3 μg/mouse) (FIG. 4). All threedoses of IL4/IL13 fusion protein markedly reduced paclitaxel-inducedpolyneuropathy. Importantly, the almost normalization of mechanicalhyperalgesia lasted for at least a week, demonstrating the potential ofthe IL4/IL13 fusion protein for long-lasting resolution ofchemotherapy-induced polyneuropathy.

Example 5 Potency of IL4/IL13 Fusion Protein is Superior Over IL4/IL10Fusion Protein or IL4 and IL13 Combination Therapy to CureChemotherapy-Induced Polyneuropathy

Next it was assessed whether IL4/IL13 fusion protein inhibitspaclitaxel-induced polyneuropathy better than IL4/IL10 fusion protein,or the combination of IL4 and IL13. Mice developed paclitaxel-inducedpainful polyneuropathy after 4 injections of paclitaxel (8 mg/kg) everyother day from day 0 to 6. IL4/IL13 fusion protein (comprising SEQ IDNO: 4) inhibited paclitaxel-induced mechanical hypersensitivity for atleast 1 week, whilst the combination of wildtype IL4 and IL13, orIL4/IL10 fusion protein only inhibited paclitaxel-induced mechanicalhypersensitivity for 1-2 days (FIG. 5). The inhibition ofpaclitaxel-induced persistent allodynia by IL4/IL13 fusion protein wasassociated with reduced paclitaxel-induced intra-epidermal nerve fibreloss in the paw skin (FIG. 6).

To evaluate whether IL4/IL13 fusion protein protects againstneurotoxicity induced by paclitaxel in vitro we measured neurite lengthof mouse sensory neurons cultured in presence of paclitaxel with orwithout fusion protein. Paclitaxel had a significant negative effect onneurite length when compared to the control group (FIG. 7). Simultaneouspresence of IL4/IL10 fusion protein or the combination of IL4 and IL13had a moderate beneficial effect on neurite length. However, presence ofIL4/IL13 fusion protein in the culture markedly preventedpaclitaxel-induced neurotoxicity (FIG. 7). Thus, these data togetherdemonstrated an unexpected superior effect of IL4/IL13 fusion proteinover IL4/IL10 fusion protein or the combination of IL4 and IL13 toprotect neurons against toxic effects of the chemotherapeutic drugpaclitaxel. In particular the superiority of the fusion protein over thecombination was surprising as the in vitro system is not affected bydifferent clearance of the proteins from the site of action. Rather thedata pointed to a unique effect of IL4/IL13 fusion protein regardingneuroprotection.

Example 6 IL4/IL13 Fusion Protein Also Cures Polyneuropathy Induced byOxaliplatin

It was investigated whether neuroprotective effects of IL4/IL13 fusionprotein are unique for paclitaxel-induced neurotoxicity or whetherneuroprotective effects are against a broader spectrum ofchemotherapy-induced polyneuropathy. Toxic neuropathy was induced inmice using a platinum-based chemotherapeutic drug, oxaliplatin. Twocycles of 5 times a daily injection of oxaliplatin, separated by 5 dayswithout intraperitoneal injection, induced mechanical allodynia thatpersisted for at least 3 weeks (FIG. 8). Intrathecal injection ofIL4/IL13 fusion protein (comprising SEQ ID NO: 4) on the second dayafter the last oxaliplatin injection reduced mechanical allodyniasignificantly for 4 days (FIG. 8). Intrathecal injection of eitherwild-type IL4 or wild-type IL13 transiently inhibitedoxaliplatin-induced mechanical allodynia for ˜1 day, which wassignificantly shorter than the effect of the IL4/IL13 fusion protein.Similarly, in vitro IL4/IL13 fusion protein protected againstoxaliplatin-induced neurotoxicity, whilst the combination of IL4 andIL13 did not (FIG. 9). Thus, these data indicate that IL4/IL13 fusionprotein protects against chemotherapy-induced neurotoxicity andchemotherapy-induced polyneuropathy.

Example 7 IL10/IL13 and IL33/IL13 Fusion Proteins

Further, it was considered whether an IL10/IL13 fusion protein orIL33/IL13 fusion protein would have a therapeutic effect in the medicalindications as disclosed herein. Surprisingly, cross-linking of the IL10receptor and the IL13 receptor by administration of an IL10/IL13 fusionprotein, or the cross-linking of the IL33 receptor and the IL13 receptorby administration of an IL33/IL13 fusion protein, can lead to a strongerand prolonged therapeutic effect, in comparison to administration of anIL4/IL10 fusion protein, and in particular in the neuropathic painmodel.

Example 8 Expression of Cytokine Receptors in Dorsal Root Ganglia andSpinal Cord

It was addressed whether cytokine receptors targeted by fusion proteinsof the present invention are expressed by the sensory system. To analyzethis, RNAseq data of receptors for IL10, IL4, IL13, IL27, TGFβ1, andTGFβ2 in the dorsal root ganglia and spinal cord were extracted from thedata base by Ray et al. (Pain 2018; 159:1325-1345) as available onwww_utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?go. RNAsequencing revealed expression of receptor chains for IL10, IL4, IL13,IL27, TGFβ1 and TGFβ2 in the dorsal root ganglia and spinal cord ofhuman and mouse (FIG. 10; data are expressed as transcripts permillion).

Example 9 Fusion Proteins of the Disclosure

This example demonstrates design and generation of non-limiting examplesof IL13-containing fusion proteins of the disclosure.

An IL4/IL13 fusion protein of the disclosure comprising SEQ ID NO:1 andSEQ ID NO: 14 was designed. SEQ ID NO: 1 was joined to SEQ ID NO: 14using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 16. Ahexa-histidine tag was added to the N-terminus, and the fusion proteinwas produced by transient transfection of HEK293E cells as disclosedbelow. The fusion protein containing SEQ ID NO: 16, has IL4 located atthe N-terminal end, and is labeled as IL4/IL13_(SKP) in FIG. 11, FIG.12, and FIG. 18A.

An IL10/IL13 fusion protein of the disclosure comprising SEQ ID NO:5 andSEQ ID NO: 14 was designed. SEQ ID NO: 5 was joined to SEQ ID NO: 14using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 17. Ahexa-histidine tag was added to the N-terminus, and the fusion proteinwas produced by transient transfection of HEK293E cells as disclosedbelow. The fusion protein containing SEQ ID NO: 17 contains IL13 at theN-terminal end and is labeled as IL13/IL10 in FIG. 11, FIG. 13, and FIG.18D.

An IL27/IL13 fusion protein of the disclosure comprising SEQ ID NO:18and SEQ ID NO: 14 was designed. SEQ ID NO: 18 was joined to SEQ ID NO:14 using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 19. Ahexa-histidine tag was added to the N-terminus, and the fusion proteinwas produced by transient transfection of HEK293E cells as disclosedbelow. The fusion protein containing SEQ ID NO: 19 contains IL13 at theN-terminal end and is labeled IL13/IL27-A in FIG. 11, FIG. 14, and FIG.18C.

An IL13/IL13 fusion protein of the disclosure comprising two copies ofSEQ ID NO: 14 was designed. SEQ ID NO: 14 was joined to a second copy ofSEQ ID NO: 14 using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 20.A hexa-histidine tag was added to the N-terminus, and the fusion proteinwas produced by transient transfection of HEK293E cells as disclosedbelow.

The IL4/IL13, IL10/IL13, IL27/IL13, and IL13/IL13 fusion proteins werepurified as disclosed below. Size exclusion chromatography indicatedthat the IL4/IL13 fusion protein containing SEQ ID NO: 16 migrated as asingle peak (FIG. 18A), and the IL13/IL13 fusion protein (containing SEQID NO: 20) migrated as a single peak (FIG. 18B).

For IL27/IL13 (IL13/IL27-A), two peaks of recombinant protein wereidentified which were separately pooled after gel filtration (FIG. 18C).Pool 1 was the higher molecular weight pool. Pool 2 was of lowermolecular weight, and was elected for further evaluation. Withoutwishing to be bound by any particular theory, pool 1 may containmultimerized and/or aggregate forms of the protein, while pool 2 maycontain a pure protein (e.g., a monomer)

For IL10/IL13 (IL13/IL10), two peaks of recombinant protein wereidentified which were separately pooled after gel filtration (FIG. 18D).Pool 1 represents the high molecular weight pool and pool 2 the lowmolecular weight pool. Without wishing to be bound by any particulartheory, IL10/IL13 pool 1 may contain a dimer version of the molecule,and IL10/IL13 pool 2 may contain a monomer version of IL10/IL13.

As shown in FIG. 11, purified proteins were analysed on 4-12% gradientpolyacrylamide NuPage™ gels under reducing and non-reducing conditions,and bands were visualized by Coomassie protein stain.

Expression and purification of IL13-containing fusion proteins.IL13-containing fusion proteins of the disclosure were produced bytransient transfection of HEK293E cells. Cells were transfected with apUPE expression vector containing a transgene coding one of theIL13-containing fusion protein sequences. To enable purification, ahexa-histidine affinity tag was cloned at the N-terminus of eachIL13-containing protein. Six days post transfection, conditioned mediumcontaining recombinant protein was harvested by low-speed centrifugation(10 minutes, 1000×g) followed by high-speed centrifugation (10 minutes,4000×g).

Proteins were purified via His-tag by Immobilized Metal AffinityChromatography (IMAC). In short, the recombinant protein was bound to0.5 ml Nickel sepharose° excel at 20° C. Nickel sepharose® excelcontaining bound protein was harvested by centrifugation and transferredinto a gravity flow column. Non-specifically bound proteins were removedby washing the column with IMAC buffer (500 mM Sodium Chloride, 25 mMTris, pH=8.2) containing 0 and 10 mM imidazol. The proteins were elutedwith IMAC buffer containing 500 mM imidazol. Fractions of 2.5 ml werecollected and recombinant protein-containing fractions were pooled.Conditioned medium and the unbound IMAC fraction were analyzed byLabChip® capillary electrophoresis. The IMAC pool was concentrated to2-4 ml using an Amicon 10 kDa spin filter. Aggregates were removed bycentrifugation (10 minutes 18000×g, 4° C.).

The proteins were purified further by gel filtration using a Superdex20016/600 column that had been equilibrated in PBS. Protein containingfractions were analyzed by LabChip® capillary electrophoresis andrecombinant protein containing fractions were pooled. Protein pools weresterilized by filtration over a 0.22 μm syringe filter and the productstored in 1 ml vials at −80° C.

Protein assays: Protein concentration was determinedspectrophotometrically by measuring the absorbance at 280 nm(DropSense16, Trinean) and using a BCA (Thermo Scientific) proteinassay. All protein assays were performed according to the manufacturer'sprotocols.

SDS-Page: Purified proteins were analysed on 4-12% polyacrylamideNuPage™ polyacrylamide gels under reducing and non-reducing conditions.Protein bands were visualized by Coomassie protein stain.

Example 10 Neuroprotective Effects of IL13-Containing Fusion Proteins onPaclitaxel-Induced Sensory Neuron Damage

To elucidate the potential neuroprotective effect of various fusionproteins where IL13 is linked to other regulatory cytokines such as IL4,IL10, IL13, or IL27, the fusion proteins were tested in an in vitropaclitaxel-induced sensory neuron damage assay. Primary sensory neuronsfrom mice were cultured for 24 h in the presence of paclitaxel (1 μM),and different concentrations of each fusion protein or equimolar dosesof IL13 or the combination of unlinked cytokines.

Culture of DRG neurons: DRGs were cultured as described previously (Nat.Commun. 4, 1682 (2013)). Briefly, DRGs were dissected and placed onice-cold dissection medium (HBSS w/o Ca²⁺ and Mg²⁺, 5 mM HEPES, and 10mM glucose). After dissection, axons were cut and dissection medium wasreplaced by filtered enzyme mix (HBSS w/o Ca²⁺ and Mg²⁺, 5 mM HEPES, 10mM glucose, 5 mg/ml collagenase type XI (Sigma), and 10 mg/ml Dispase(Gibco)). The DRGs were incubated in enzyme mix for 30 minutes at 37° C.and 5% CO₂. Subsequently, enzyme mix was inactivated withheat-inactivated fetal bovine serum (FBS, Sigma). Cells were cultured inDulbecco's modified Eagle's medium (Gibco) containing 10% FBS (Gibco), 2mmol/L glutamine (Gibco), 10,000 IU/ml penicillin-streptomycin (Gibco)on poly-L-lysine (0.01 mg/ml, Sigma) and laminin (0.02 mg/ml,Sigma)-coated glass coverslips in a 5% CO₂ incubator at 37° C. Cellswere used the following 1-2 days.

Stimulation for neurite length measurement: After 24 h in culture, DRGneurons were treated with Paclitaxel (1 μM) alone (n=11) or in presenceof different concentrations of IL13 fusion proteins (0.12 nM, n=6; 0.6nM or 3 nM, n=11) or equimolar doses of the individual cytokines (n=6)for 24 h (n-values represent the number of animals for which eachcondition was tested). Neurites were visualised using β3-tubulinstaining, whilst number of sensory neurons were determined using NeuNstaining. For each animal, 2-3 wells were evaluated (cytokines: 2 wellsper concentration; paclitaxel or control: 3 wells) and 5 pictures perwell were taken. For each picture, the neurite length per sensory neuronwas determined and averaged to a single value per animal and condition.The percentage inhibition of paclitaxel-induced neurite length loss permouse culture of 1 mouse was calculated according the following formula((μ_(control)−μ_(paclitaxel))−(μ_(control)−X_(cytokine)))/(μ_(control)−μ_(paclitaxel))*100(where μ is the neurite length per neuron averaged over all samples andX is the neurite length of each individual sample).

β3-tubulin and NeuN staining: cells were fixed in 4% PFA for 10 minutes,permeabilized with PBS with 0.05% Tween-20, followed by incubation inblocking buffer (1% BSA and 5% Normal donkey serum in PBS with 0.05%Tween-20 and 0.01% triton) for 1 hour. Cells were incubated with rabbitanti-β3 tubulin (Anti-beta III Tubulin antibody-Neuronal Marker,ab18207, 1:1500, Abcam, Cambridge, UK) and NeuN (Anti-NeuN Antibodyclone A60, MAB377, 1:500) Sigma Aldrich (Merck), Darmstadt, Germany)overnight at 4° C., followed by washes and incubation withAF488-conjugated donkey anti-rabbit and 568-conjugated donkey anti-mousesecondary antibodies (Thermofisher, 1:500) followed by DAPI (1:5000,Sigma) staining before sections were mounted on slides with FluorSavereagent (Millipore).

Images were taken using an Olympus IX83 microscope (Olympus). Pictureswere analysed using CellSens software (Olympus) and ImageJ (NIH), usingthe NeuralNetrics macro (Pani G, et al. MorphoNeuroNet: An automatedmethod for dense neurite network analysis. Cytom Part A. 2014 February;85(2):188-99). Other plugins used were Olympus Viewer plugin (Olympus).Cell sense software was used to automatically count number of neuronsbased on NeuN staining. Neurite length was determined using theNeuralNetric macro in ImageJ (Fiji).

Results

Paclitaxel reduced neurite length with approximately 67% compared tocontrol cultured sensory neurons.

FIG. 12 illustrates a comparison of the neuroprotective effect of twoIL4/IL13 fusion proteins versus IL13 alone or the combination of IL4 andIL13. IL13 dose dependently inhibited the paclitaxel-induced decrease inneurite length, with a maximum of ˜9% at a concentration of 3 nM (FIG.12). The combination of IL4 and IL13 did not significantly inhibitpaclitaxel-induced neurite length at any concentration tested.

The fusion protein labeled IL4/IL13 comprises SEQ ID NO: 4. The fusionprotein labeled IL4/IL13_(SKP) comprises SEQ ID NO: 16. IL13 is locatedat the C-terminal end of these constructs. IL4/IL13 dose-dependentlyinhibited the paclitaxel-induced reduction in neurite length, with amaximum effect of 35% at 3 nM (FIG. 12). IL4/IL13 inhibitedpaclitaxel-induced reduction in neurite length significantly better thanIL4 and IL13 combined or IL13 alone, with ˜4 fold more inhibition at aconcentration of 3 nM compared to IL13 (FIG. 12). An alternativeIL4/IL13 fusion protein, IL4/IL13_(SKP), inhibited paclitaxel-inducedreduction in neurite length to a similar extent as IL4/IL13 at allconcentrations tested (FIG. 12). The IL13 protein sequence (SEQ ID NO:14) used in IL4/IL13_(SKP) (SEQ ID NO: 16) contains an additional serineN-terminally, and an arginine instead of glutamine near the C-terminalend (position 112 in SEQ ID NO: 14). Despite these protein sequencedifferences, both fusion proteins comprising IL4 and IL13 show thesuperior neuroprotective effect versus the individual IL4 and IL13 aswell as the combination of IL4+IL13. Data are shown as mean±SEM. Dataare analysed with a two-way ANOVA mixed-effects analysis followed byTukey's multiple comparison test.

FIG. 13 illustrates a comparison of neuroprotective effects of two poolsof IL10/IL13 fusion proteins to equimolar concentrations of IL13 alone,or the combination of IL10 and IL13. The IL10/IL13 fusion proteinscomprise SEQ ID NO: 17, with IL13 located at the N-terminal end. Poolsof this fusion protein are referred to as IL13/IL10_(pool1) andIL13/IL10_(pool2) in the figure. IL13/IL10_(pool1) significantlyinhibited paclitaxel-induced reduction in neurite length at all dosestested, ranging from 15% at 0.12 nM to 28% at 3 nM (FIG. 13).IL13/IL10_(pool2) also concentration-dependently protected neurons frompaclitaxel-induced reduction in neurite length. Importantly bothIL13/IL10_(pool1) and IL13/IL10_(pool2) prevented paclitaxel-inducedneuronal damage significantly better than IL13 or the combination ofIL10 and IL13 at 3 nM (FIG. 13). Data are shown as mean±SEM. Data areanalysed with a two-way ANOVA mixed-effects analysis followed by Tukey'smultiple comparison test.

FIG. 14 provides a comparison of neuroprotective effects of an IL13/IL13fusion protein and an IL27/IL13 fusion protein to IL13 alone. TheIL13/IL13 fusion protein comprises SEQ ID NO: 20. The IL13/IL13 fusionprotein inhibited paclitaxel-induced reduction in neurite length by ˜10%at 0.12 nM, which was significantly better than IL13 at thatconcentration. However, with increasing concentration IL13/IL13 did notprovide more neuroprotection (FIG. 14).

The IL27/IL13 fusion protein comprises SEQ ID NO: 19, with IL13 locatedat the N terminal end of the protein, and is referred to asIL13/IL27-A_(pool2) in FIG. 14 This fusion protein includes IL13 and asecretion-competent mutein of IL27A (Müller et al., 2019). Pool 2 ofIL27/IL13, generated as described above, was tested. At 0.6 nM,IL27/IL13 inhibited paclitaxel-induced neuronal damage with a maximaleffect of ˜21% inhibition. At this concentration, IL27/IL13 outperformedIL13. At 3 nM the neuroprotective effects of IL13 and IL27/IL13 were notsignificantly different. Data are shown as mean±SEM. Data are analysedwith a two-way ANOVA mixed-effects analysis followed by Tukey's multiplecomparison test.

These data demonstrate that multiple fusion proteins comprising IL13 anda regulatory cytokine are effective in reducing neurotoxicity, includingfusion proteins comprising different IL13 sequences, differentregulatory cytokines, and with IL13 at either the N-terminal orC-terminal end of the fusion protein. Multiple fusion proteinsdemonstrate superiority over IL13 alone, and superiority over thecombination of IL13 and the regulatory cytokine.

Example 11 An IL13-Containing Fusion Protein of the Disclosure Elicits aDistinct Kinase Activity Profile Compared to a Combination of UnlinkedCytokines

Animals: All animal experiments were performed in accordance withinternational guidelines and with prior approval from the UniversityMedical Center Utrecht experimental animal committee. Experiments wereconducted with 8-14 weeks old male and female wild type (WT) C57BL/6mice.

Paclitaxel-induced CIPN: At day 0, 2, 4 and 6 animals were injectedintraperitoneally with 8 mg/kg of Paclitaxel (diluted in Cremophor:EtOH1:1; volume of injection 40 μl/10 g of bodyweight. At day 8, animalsreceived i.t. injections of: IL4/IL13 fusion protein (0.3 μg), IL4+IL13(0.15 μg each) or Vehicle. The IL4/IL13 fusion protein was a fusionprotein comprising SEQ ID NO: 4.

Drugs and administration: The IL4/IL13 was produced by transienttransfection of HEK293F cells with the pcDNA3.1-neo expression vector(Invitrogen; Carlsbad, Calif.) with dual CMV promotor. The vectorcontained two transgenes: cDNA coding for IL4/IL13 fusion protein andcDNA coding beta-galactoside-2, 3-sialyl-transferase to optimize glycancapping with sialic acid. The IL4/IL13 contained a 6-His tag at the Nterminus and was purified through HIS-Select Nickel Affinity gel(Sigma). IL4/IL13 concentrations were determined with an IL4 ELISA kit(IL-4 Pelipair ELISA kit; Sanquin) and Bicinchoninic Acid Protein Assay(BCA Pierce Protein Assay Kit, ThermoFisher Scientific). Intrathecal(i.t.) injections of different compounds (5 μl/mouse) were performed asdescribed before (J Neurosci 30, 2138-2149, 2010) under lightisoflurane/O₂ anesthesia. The IL4/IL13 (0.3 μg/mouse) or equimolar doses(0.15 μg each/mouse) of recombinant human HEK-produced IL4 (Sigma) andIL13 (2bsciences) were injected intrathecally at day 8 after the firstpaclitaxel injection.

Kinase Activity Profiling: Animals were killed an hour after intrathecalinjection of the IL4/IL13 fusion protein, IL4+IL13 or vehicle, followedby immediate DRG isolation. Lumbar DRGs were homogenized using M-PERmammalian Extraction buffer (Pierce) supplemented with phosphatase andprotease inhibitor cocktails (Pierce). Protein concentration wasdetermined using the Bradford assay (Bio-Rad). Kinase activity profilingwas performed using the Tyrosine Kinase PamChip® (PTK) Array forPamstation®12 (PamGene International B.V.). For the PTK array, 7.5 μg ofprotein lysate per array was used. Image quantification and statisticalanalysis were performed using BioNavigator® Software (PamGeneInternational B.V.). Upstream kinase analysis was performed usingBioNavigator® Software with peptide-kinase mapping using Kinexusphosphonet enrichment files (www_phosphonet.ca/).

Results

To elucidate downstream signaling in sensory neurons in an unbiasedmanner, PamGene kinase activity profiling was performed to assess globalprotein tyrosine kinases (PTK) activity in homogenates of lumbar DRGsisolated from mice with persistent paclitaxel-induced CIPN afterIL4/IL13 fusion protein, IL4+IL13 (combination of unlinked cytokines),and vehicle administration. Kinomic profiles were assessed at 60 minutesafter intrathecal administration of the IL4/IL13 fusion protein, thecombination of cytokines, or vehicle (PBS). Naive mice (i.e. not treatedwith paclitaxel or IL4/IL13 fusion protein) were also included.

FIG. 15 illustrates peptides that were differentially phosphorylatedbased on one-way ANOVA analysis between IL4/IL13, IL4+IL13, andvehicle-treated mice compared to naive mice (untreated; no paclitaxel,no intrathecal injection). Black indicates no significant changes, whilecolor indicates decreased phosphorylation. Analyses of the peptides thatwere differentially phosphorylated by PTK in the DRG homogenates ofIL4/IL13-treated versus IL4 plus IL13-treated mice and vehicle-treatedmice, indicated that in total 19 peptides were uniquely phosphorylatedupon treatment with the fusion protein.

Analyses of the peptides that were differentially phosphorylated by PTKin the DRG homogenates of IL4/IL13-treated male and female mice versusIL4 plus IL13-treated mice, indicated that in both sexes the activity ofdifferent kinases is differentially affected by the IL4/IL13 compared tothe combination of cytokines (FIG. 16 and FIG. 17).

In DRGs from female mice treated with the IL4/IL13 fusion protein, theactivity of several kinases was reduced when compared female micetreated with the combination of IL4 and IL13 (FIG. 16). The graph showsthe predicted upstream kinases inferred from the differentiallyphosphorylated peptide substrates on the PamChips® identified byunpaired t-test comparison between samples from IL4/IL13 fusionprotein-treated females and IL4+IL13-treated females (n=3 animals pergroup).The top 5 predicted putative kinases affected (according tosummation of sensitivity and specificity score) were: ITK, RET, TYK2,FER and ERBB4.

In male mice, unique kinase activity was predominantly increased (FIG.17). The graph shows predicted upstream kinases that can be inferredfrom the differentially phosphorylated peptide substrates on thePamChips® identified by unpaired t-test comparison between samples fromIL4/IL13 fusion protein-treated males and IL4+IL13-treated males (n=3animals per group). The top 5 predicted putative kinases affected were:LTK, RYK, ALK, AXL and BLK.

These data show that IL4/IL13 uniquely regulates sets of kinasescompared to the combination of unlinked IL4 plus IL13.

Example 12 Additional Fusion Proteins of the Disclosure

This example demonstrates design and generation of non-limiting examplesof IL13-containing fusion proteins of the disclosure.

An IL33/IL13 fusion protein of the disclosure comprising SEQ ID NO: 6and SEQ ID NO: 14 is designed. SEQ ID NO: 6 is joined to SEQ ID NO: 14using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 23. The fusionprotein is produced as disclosed herein. For example, a hexa-histidinetag is added to the N-terminus, and the fusion protein is produced bytransient transfection of HEK293E cells as disclosed below.

A TGFβ1/IL13 fusion protein of the disclosure comprising SEQ ID NO: 21and SEQ ID NO: 14 is designed. SEQ ID NO: 21 is joined to SEQ ID NO: 14using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 24. The fusionprotein is produced as disclosed herein. For example, a hexa-histidinetag is added to the N-terminus, and the fusion protein is produced bytransient transfection of HEK293E cells as disclosed below.

A TGFβ2/IL13 fusion protein of the disclosure comprising SEQ ID NO: 22and SEQ ID NO: 14 is designed. SEQ ID NO: 22 is joined to SEQ ID NO: 14using the SEQ ID NO: 3 linker, resulting in SEQ ID NO: 25. The fusionprotein is produced as disclosed herein. For example, a hexa-histidinetag is added to the N-terminus, and the fusion protein is produced bytransient transfection of HEK293E cells as disclosed below.

Additional IL4/IL13 fusion proteins are designed wherein any one of SEQID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to any one of SEQ ID NOs: 1 or 26-28 (or a variant, derivative,or fragment thereof), either directly or via a linker as disclosedherein (for example, any one of SEQ ID NOs: 3 or 37-44, or a multiplethereof). The fusion proteins are designed in both orientations, e.g.,with IL4 located on the C-terminal side of IL13, or with IL4 located onthe N-terminal side of IL13. The fusion proteins are produced asdisclosed herein. For example, an affinity tag is added to theN-terminus and/or the C-terminus of each fusion protein, and the fusionproteins are produced by transient transfection of HEK293E cells asdisclosed below.

Additional IL10/IL13 fusion proteins are designed wherein any one of SEQID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to SEQ ID NOs: 5 (or a variant, derivative, or fragment thereof),either directly or via a linker as disclosed herein (for example, anyone of SEQ ID NOs: 3 or 37-44, or a multiple thereof). The fusionproteins are designed in both orientations, e.g., with IL10 located onthe C-terminal side of IL13, or with IL10 located on the N-terminal sideof IL13. The fusion proteins are produced as disclosed herein. Forexample, an affinity tag is added to the N-terminus and/or theC-terminus of each fusion protein, and the fusion proteins are producedby transient transfection of HEK293E cells as disclosed below.

Additional IL27/IL13 fusion proteins are designed wherein any one of SEQID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to any one of SEQ ID NOs: 18, 36, 45, or a combination thereof(or a variant, derivative, or fragment thereof), either directly or viaa linker as disclosed herein (for example, any one of SEQ ID NOs: 3 or37-44, or a multiple thereof). The fusion proteins are designed in bothorientations, e.g., with IL27 located on the C-terminal side of IL13, orwith IL27 located on the N-terminal side of IL13. The fusion proteinsare produced as disclosed herein. For example, an affinity tag is addedto the N-terminus and/or the C-terminus of each fusion protein, and thefusion proteins are produced by transient transfection of HEK293E cellsas disclosed below.

Additional IL33/IL13 fusion proteins are designed wherein any one of SEQID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to any one of SEQ ID NOs: 6 or 29 or 34 (or a variant,derivative, or fragment thereof), either directly or via a linker asdisclosed herein (for example, any one of SEQ ID NOs: 3 or 37-44, or amultiple thereof). The fusion proteins are designed in bothorientations, e.g., with IL33 located on the C-terminal side of IL13, orwith IL33 located on the N-terminal side of IL13. The fusion proteinsare produced as disclosed herein. For example, an affinity tag is addedto the N-terminus and/or the C-terminus of each fusion protein, and thefusion proteins are produced by transient transfection of HEK293E cellsas disclosed below.

Additional IL13/IL13 fusion proteins are designed wherein any one of SEQID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to any one of SEQ ID NOs: 2 or 9-15 (or a variant, derivative, orfragment thereof), either directly or via a linker as disclosed herein(for example, any one of SEQ ID NOs: 3 or 37-44, or a multiple thereof).The fusion proteins are designed in both orientations, e.g., with thefirst IL13 located on the C-terminal side of the second IL13, or withfirst IL13 located on the N-terminal side of second IL13. The first IL13and the second IL13 can be the same or different. The fusion proteinsare produced as disclosed herein. For example, an affinity tag is addedto the N-terminus and/or the C-terminus of each fusion protein, and thefusion proteins are produced by transient transfection of HEK293E cellsas disclosed below.

Additional TGFβ1/IL13 fusion proteins are designed wherein any one ofSEQ ID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to any one of SEQ ID NOs: 7 or 21 (or a variant, derivative, orfragment thereof), either directly or via a linker as disclosed herein(for example, any one of SEQ ID NOs: 3 or 37-44, or a multiple thereof).The fusion proteins are designed in both orientations, e.g., with TGFβ1located on the C-terminal side of IL13, or with TGFβ1 located on theN-terminal side of IL13. The fusion proteins are produced as disclosedherein. For example, an affinity tag is added to the N-terminus and/orthe C-terminus of each fusion protein, and the fusion proteins areproduced by transient transfection of HEK293E cells as disclosed below.

Additional TGFβ2/IL13 fusion proteins are designed wherein any one ofSEQ ID NOs: 2 or 9-15 (or a variant, derivative, or fragment thereof) isjoined to any one of SEQ ID NOs: 8, 22, or 35 (or a variant, derivative,or fragment thereof), either directly or via a linker as disclosedherein (for example, any one of SEQ ID NOs: 3 or 37-44, or a multiplethereof). The fusion proteins are designed in both orientations, e.g.,with TGFβ2 located on the C-terminal side of IL13, or with TGFβ2 locatedon the N-terminal side of IL13. The fusion proteins are produced asdisclosed herein. For example, an affinity tag is added to theN-terminus and/or the C-terminus of each fusion protein, and the fusionproteins are produced by transient transfection of HEK293E cells asdisclosed below.

IL13-containing fusion proteins of the disclosure are produced bytransient transfection of HEK293E cells. Cells are transfected with apUPE expression vector containing a transgene coding one of theIL13-containing fusion protein sequences. To enable purification, ahexa-histidine affinity tag is cloned at the N-terminus of eachIL13-containing protein. Six days post transfection, conditioned mediumcontaining recombinant protein is harvested by low-speed centrifugation(10 minutes, 1000×g) followed by high-speed centrifugation (10 minutes,4000×g).

Proteins are purified via His-tag by Immobilized Metal AffinityChromatography (IMAC). In short, the recombinant protein is bound to 0.5ml Nickel sepharose® excel at 20° C. Nickel sepharose® excel containingbound protein is harvested by centrifugation and transferred into agravity flow column. Non-specifically bound proteins are removed bywashing the column with IMAC buffer (500 mM Sodium Chloride, 25 mM Tris,pH=8.2) containing 0 and 10 mM imidazol. The proteins are eluted withIMAC buffer containing 500 mM imidazol. Fractions of 2.5 ml arecollected. Recombinant protein-containing fractions are pooled.Conditioned medium and the unbound IMAC fraction are analyzed byLabChip® capillary electrophoresis. The IMAC pool is concentrated to 2-4ml using an Amicon 10 kDa spin filter. Aggregates are removed bycentrifugation (10 minutes 18000×g, 4° C.).

The proteins are purified further by gel filtration using a Superdex20016/600 column that has been equilibrated in PBS. Protein containingfractions are analyzed by LabChip® capillary electrophoresis andrecombinant protein containing fractions are pooled. Protein pools aresterilized by filtration using a 0.22 μm syringe filter and the productstored in 1 ml vials at −80° C.

Protein assays: Protein concentration in batches is determinedspectrophotometrically by measuring the absorbance at 280 nm(DropSense16, Trinean) and using a BCA (Thermo Scientific) proteinassay.

SDS-Page: Purified proteins are analysed on 4-12% polyacrylamide NuPage™polyacrylamide gels under reducing and non-reducing conditions. Proteinbands are visualized by Coomassie protein stain.

Example 13 Neuroprotective Effects of Fusion Proteins of the Disclosure

Assays are conducted to assess whether the IL13-containing fusionproteins possess neuroprotective properties, e.g., inhibitpaclitaxel-induced reduction of neurite length. The assays are conductedfor any fusion protein disclosed herein, for example, fusion proteinscomprising an IL13 and a regulatory cytokine, e.g., an IL4/IL13,IL10/IL13, IL13/IL13, IL27/IL13, IL33/IL13, TGFβ1/IL13, or TGFβ2/IL13 ofthe disclosure.

Culture of DRG neurons: DRGs are cultured as described previously (Nat.Commun. 4, 1682 (2013)). Briefly, DRGs are dissected and placed onice-cold dissection medium (HBSS w/o Ca²⁺ and Mg²⁺, 5 mM HEPES, and 10mM glucose). After dissection, axons are cut and dissection medium isreplaced by filtered enzyme mix (HBSS without Ca²⁺ and Mg²⁺, 5 mM HEPES,10 mM glucose, 5 mg/ml collagenase type XI (Sigma), and 10 mg/ml Dispase(Gibco)). The DRGs are incubated in enzyme mix for 30 minutes at 37° C.and 5% CO₂. Subsequently, enzyme mix is inactivated withheat-inactivated fetal bovine serum (FBS, Sigma). Cells are cultured inDulbecco's modified Eagle's medium (Gibco) containing 10% FBS (Gibco), 2mmol/L glutamine (Gibco), 10,000 IU/ml penicillin-streptomycin (Gibco)on poly-L-lysine (0.01 mg/ml, Sigma) and laminin (0.02 mg/ml,Sigma)-coated glass coverslips in a 5% CO₂ incubator at 37° C. Cells areused the following 1-2 days.

Treatments and neurite length measurement: After 24 h in culture, DRGneurons are treated with Paclitaxel (1 μM) alone, or in the presence ofdifferent concentrations of IL13 fusion proteins (e.g., 0.12 nM, 0.6 nM,or 3 nM), or equimolar doses of the individual cytokines that arepresent in the fusion proteins for 24 h. Neurites are visualised usingβ3-tubulin staining, whilst the number of sensory neurons is determinedusing NeuN staining. Pictures are taken and for each picture, theneurite length per sensory neuron is determined. The neurite length isaveraged to a single value for each animal and condition. The percentageinhibition of paclitaxel-induced neurite length loss per mouse cultureof 1 mouse is calculated according the following formula((μ_(control)−μ_(paclitaxel))−(μ_(control)−X_(cytokine)))/(μ_(control)−μ_(paclitaxel))*100(where μ is the neurite length per neuron averaged over all samples andX is the neurite length of each individual sample).

β3-tubulin and NeuN staining: cells are fixed in 4% PFA for 10 minutes,permeabilized with PBS with 0.05% Tween-20, followed by incubation inblocking buffer (1% BSA and 5% Normal donkey serum in PBS with 0.05%Tween-20 and 0.01% triton) for 1 hour. Cells are incubated with rabbitanti-β3 tubulin (Anti-beta III Tubulin antibody-Neuronal Marker,ab18207, 1:1500, Abcam, Cambridge, UK) and NeuN (Anti-NeuN Antibodyclone A60, MAB377, 1:500) Sigma Aldrich (Merck), Darmstadt, Germany)overnight at 4° C., followed by washes and incubation withAF488-conjugated donkey anti-rabbit and 568-conjugated donkey anti-mousesecondary antibodies (Thermofisher, 1:500) followed by DAPI (1:5000,Sigma) staining before sections are mounted on slides with FluorSavereagent (Millipore).

Images are taken using an Olympus IX83 microscope (Olympus). Picturesare analysed using CellSens software (Olympus) and ImageJ (NIH), usingthe NeuralNetrics macro (Pani G, et al. MorphoNeuroNet: An automatedmethod for dense neurite network analysis. Cytom Part A. 2014 February;85(2):188-99). Other plugins used are Olympus Viewer plugin (Olympus).Cell sense software is used to automatically count number of neuronsbased on NeuN staining. Neurite length is determined using theNeuralNetric macro in ImageJ (Fiji). The ability of IL13-containingfusion proteins of the disclosure to inhibit paclitaxel-inducedreduction of neurite length is determined and compared to thecombination of unlinked cytokines for each fusion protein.

Example 14 In Vivo Assessment of Fusion Proteins of the Disclosure

Assays are conducted to assess whether the IL13-containing fusionproteins can treat, for example, pain, chemotherapy-inducedpolyneuropathy (CIPN), pain, nerve fiber loss, and allodynia. The assaysare conducted for any fusion protein disclosed herein, for example,fusion proteins comprising an IL13 and a regulatory cytokine, e.g., anIL4/IL13, IL10/IL13, IL13/IL13, IL27/IL13, IL33/113, TGFβ1/IL13, orTGFβ2/IL13 of the disclosure.

To induce transient chemotherapy-induced polyneuropathy (CIPN),paclitaxel (2 mg/kg, Cayman Chemical Company) is injectedintraperitoneally into C57BL/6 mice on days 0 and 2. To inducepersistent paclitaxel-induced CIPN, paclitaxel (8 mg/kg, Cayman ChemicalCompany) is injected intraperitoneally on day 0, 2, 4 and 6. To inducepersistent oxaliplatin-induced polyneuropathy, mice receive twotreatment cycles, each consisting of 5 daily intraperitoneal injectionsof 3 mg/kg oxaliplatin (Tocris) with a 5 days free interval. To induceinflammatory hyperalgesia, mice receive an intraplantar injection of 20μl λ-carrageenan (2% (w/v), Sigma-Aldrich) dissolved in saline solution(NaCl 0.9%) in both hind paws. In other animals, chronic constrictioninjury (CCI) and spared nerve injury (SNI) models are used.

Noxious mechanical sensitivity in the hind paws is measured using vonFrey hairs (Stoelting, Wood Dale, USA). Results are expressed as the 50%paw-withdrawal threshold using the up-and-down method. Thermalhyperalgesia is assessed by determining the heat withdrawal latencytimes using the Hargreaves test (IITC Life Science). In some experimentsthe length of intraepidermal nerve fibers in the paw skin at day 15 isdetermined by immunofluorescent staining of skin biopsies with theneuronal marker PGP9.5. All experiments are performed in a blindedmanner.

IL13-containing fusion proteins are administered to mice, e.g., viaintrathecal injection under light isoflurane/O₂ anaesthesia, or byanother route as disclosed herein. The ability of IL13-containing fusionproteins of the disclosure to inhibit neuropathy, hyperalgesia, andintra-epidermal nerve fibre loss, is determined and compared to thecombination of unlinked cytokines for each fusion protein.

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SEQUENCE LISTING SEQ ID NO: 1 Amino acid sequence of human IL4HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAA SKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQ STLENFLERLKTIMREKYSKCSS SEQ ID NO: 2Amino acid sequence of human IL13 PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGF CPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN SEQ ID NO: 3 Amino acid sequence of suitable linker GSGGGGSGTAmino acid sequence of an IL4/1L13 fusion protein (the linker sequenceis underlined SEQ ID NO: 4 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATA QQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTPGP VPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPH KVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN SEQ ID NO: 5 Amino acid sequence of human IL10SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKT FFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRL RRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 6 Amino acid sequence of human IL33MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQK AKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYT RALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGD GVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDN HLALIKVDSSENLCTENILFKLSETSEQ ID NO: 7 Amino acid sequence of human immatureand mature (underlined) TGFβ1: MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSEL REAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSR GGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRAL DTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGA SAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS SEQ ID NO: 8 Amino acid sequence of human immatureand mature (underlined) TGFβ2: MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISI YNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSETVCPVVTTPSGSVGSLCSRQSQVLC GYLDAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQ RYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAG IDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRP YIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILY YIGKTPKIEQLSNMIVKSCKCS SEQ ID NO: 9Amino acid sequence of human IL13 GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFC PHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN Amino acid sequence of human IL13 SEQ ID NO: 10SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMV WSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLK KLFREGQFN SEQ ID NO: 11Amino acid sequence of human IL13 LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIE KTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN SEQ ID NO: 12 Amino acid sequence of human IL13PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVW SINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKK LFREGRFN SEQ ID NO: 13Amino acid sequence of human IL13 GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFC PHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN Amino acid sequence of human IL13 SEQ ID NO: 14SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMV WSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLK KLFREGRFN SEQ ID NO: 15Amino acid sequence of human IL13 LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIE KTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN SEQ ID NO: 16 Amino acid sequence of an IL4/1L13fusion protein HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATA QQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTSPG PVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCP HKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN SEQ ID NO: 17 Amino acid sequence of IL10/1L13 fusion proteinSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMV WSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLK KLFREGRFNGSGGGGSGTSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESL LEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFlNYIEAYMTMKIRN SEQ ID NO: 18Amino acid sequence of IL27A^(L162c) FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWR RLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNCPEEEEE EEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPT LSPQP SEQ ID NO: 19Amino acid sequence of IL27/1L13 fusion proteinSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMV WSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLK KLFREGRFNGSGGGGSGTFPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYL LPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRH LRFQVLAAGFNCPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRE LLLLSKAGHSVWPLGFPTLSPQPSEQ ID NO: 20 Amino acid sequence of IL13/1L13 fusion proteinSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMV WSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLK KLFREGRFNGSGGGGSGTSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESL INVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN SEQ ID NO: 21Amino acid sequence of mature TGFβ1 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNP GASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS SEQ ID NO: 22 Amino acid sequence of mature TGFβ2ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHE PKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCS SEQ ID NO: 23Amino acid sequence of IL33/1L13 fusion proteinMKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQK AKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYT RALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGD GVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDN HLALIKVDSSENLCTENILFKLSETGSGGGGSGTSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVW SINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKK LFREGRFN SEQ ID NO: 24Amino acid sequence of TGFβ1/1L13 fusion proteinALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHE PKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMI VRSCKCSGSGGGGSGTSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLIN VSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN SEQ ID NO: 25:Amino acid sequence of TGFβ2/IL13 fusion proteinALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHE PKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCSGSGGGGSGTSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLIN VSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN SEQ ID NO: 26 Amino acid sequence of human IL4HKCDITLQEIIKTLNSLTEQKNTTEKETFCRAATV LRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMR EKYSKCSS SEQ ID NO: 27Amino acid sequence of human IL4 HKRDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATA QQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS SEQ ID NO: 28 Amino acid sequence of human IL4HKRDITLQEIIKTLNSLTEQKNTTEKETFCRAATV LRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMR EKYSKCSS SEQ ID NO: 29Amino acid sequence of human IL33 AFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYY ESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKT DPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET SEQ ID NO: 30 Amino acid sequence of human IL33SGVQKYTRALHDSSITGISPITEYLASLSTYNDQS ITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSV ELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET SEQ ID NO: 31Amino acid sequence of human IL33 HDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVD GKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLA LIKVDSSENLCTENILFKLSET SEQ ID NO: 32Amino acid sequence of human IL33 MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSL KTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITDKVLLSYYESQHPSNESGDGVDGKML MVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKV DSSENLCTENILFKLSET SEQ ID NO: 33Amino acid sequence of human IL33 MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSL KTGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKML MVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKV DSSENLCTENILFKLSET SEQ ID NO: 34Amino acid sequence of human IL33 MKPKMKYSTNKISTAKWKNTASKALCFKLGNKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHA NNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFK LSET SEQ ID NO: 35Amino acid sequence of immature TGFβ2MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRK RIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSK DLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEE LEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQD NCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDL EPLTILYYIGKTPKIEQLSNMIVKSCKCSSEQ ID NO: 36 Amino acid sequence of IL27AFPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRG QAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNME RMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLST YRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP SEQ ID NO: 37 Linker sequence GGGS SEQ ID NO: 38 Linker sequenceGGGGS SEQ ID NO: 40 Linker sequence KESGSVSSEQLAQFRSLD SEQ ID NO: 41Linker sequence EGKSSGSGSESKST SEQ ID NO: 42 Linker sequenceGSAGSAAGSGEF SEQ ID NO: 43 Linker sequence EAAAK SEQ ID NO: 44Linker sequence EAAAR SEQ ID NO: 45 amino acid sequence of IL27BRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPN STSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEH IIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRP RARYYVQVAAQDLTDYGELSDWSLPATATMSLGK

What is claimed is:
 1. A fusion protein comprising an interleukin 13(IL13) amino acid sequence and a regulatory cytokine amino acidsequence, for use in treatment of neuropathy in a subject in needthereof.
 2. A fusion protein comprising an interleukin 13 (IL13) aminoacid sequence and a regulatory cytokine amino acid sequence, for use intreatment of pain in a subject in need thereof.
 3. A fusion proteincomprising an interleukin 13 (IL13) amino acid sequence and a regulatorycytokine amino acid sequence, for use in treatment of neurodegenerationor neuroinflammation in a subject in need thereof.
 4. A fusion proteincomprising an interleukin 13 (IL13) amino acid sequence and a regulatorycytokine amino acid sequence, for use in treatment of inflammation in asubject in need thereof.
 5. A fusion protein comprising an interleukin13 (IL13) amino acid sequence and a regulatory cytokine amino acidsequence, for use in promoting neuroprotection in a subject in needthereof.
 6. A fusion protein comprising an interleukin 13 (IL13) aminoacid sequence and a regulatory cytokine amino acid sequence, for use inmodulating activity of a signaling pathway in a nervous system cell. 7.The fusion protein for use according to any one of claims 1-6, whereinthe regulatory cytokine is selected from the group consisting of aninterleukin 4 (IL4), an interleukin 10 (IL10), an interleukin 33 (IL33),a transforming growth factor beta 1 (TGFβ1), a transforming growthfactor beta 2 (TGFβ2), and an additional interleukin 13 (IL13).
 8. Thefusion protein for use according to any one of claims 1-6, wherein theregulatory cytokine is IL4.
 9. The fusion protein for use according toany one of claims 1-6, wherein the regulatory cytokine is IL10.
 10. Thefusion protein for use according to any one of claims 1-6, wherein theregulatory cytokine is IL33.
 11. The fusion protein for use according toany one of claims 1-6, wherein the regulatory cytokine is an interleukin27 (IL27).
 12. The fusion protein for use according to any one of claims1-6, wherein the regulatory cytokine is TGFβ1.
 13. The fusion proteinfor use according to any one of claims 1-6, wherein the regulatorycytokine is TGFβ2.
 14. The fusion protein for use according to any oneof claims 1-6, wherein the regulatory cytokine is an additional IL13.15. The fusion protein for use according to any one of claims 1-14,wherein the IL13 comprises a wild type IL13.
 16. The fusion protein foruse according to any one of claims 1-15, wherein the IL13 is a mammalianIL13.
 17. The fusion protein for use according to any one of claims1-16, wherein the IL13 is a human IL13.
 18. The fusion protein for useaccording to any one of claims 1-17, wherein the regulatory cytokinecomprises a wild type regulatory cytokine.
 19. The fusion protein foruse according to any one of claims 1-18, wherein the regulatory cytokineis a mammalian regulatory cytokine.
 20. The fusion protein for useaccording to any one of claims 1-19, wherein the regulatory cytokine isa human regulatory cytokine.
 21. The fusion protein for use according toany one of claims 11-20, wherein the interleukin 27 comprises aninterleukin 27 alpha (IL27A).
 22. The fusion protein for use accordingto any one of claims 11-21, wherein the IL27A comprises an L134Csubstitution relative to SEQ ID NO:
 36. 23. The fusion protein for useaccording to any one of claims 1-22, wherein the IL13 binds tointerleukin 13 receptor alpha 1 (IL-13Rα1) with an affinity that is lessthan two fold increased and less than two fold decreased compared to awild type IL13.
 24. The fusion protein for use according to any one ofclaims 1-23, wherein the IL13 binds to interleukin 13 receptor alpha 2(IL-13Rα2) with an affinity that is less than two fold increased andless than two fold decreased compared to a wild type IL13.
 25. Thefusion protein for use according to any one of claims 1-24, wherein theIL13 binds to an interleukin 4 receptor alpha (IL-4Rα) with an affinitythat is less than two fold increased and less than two fold decreasedcompared to a wild type IL13.
 26. The fusion protein for use accordingto any one of claims 1-25, wherein the regulatory cytokine amino acidsequence is a derivative sequence that binds to all subunits of areceptor of the regulatory cytokine with a comparable affinity as a wildtype regulatory cytokine.
 27. The fusion protein for use according toany one of claims 1-26, wherein the regulatory cytokine amino acidsequence is a derivative sequence that activates a native receptor ofthe regulatory cytokine.
 28. The fusion protein for use according to anyone of claims 1-27, wherein the IL13 comprises an amino acid sequencewith at least 90% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15. 29.The fusion protein for use according to any one of claims 1-27, whereinthe IL13 comprises an amino acid sequence that is selected from thegroup consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15. 30.The fusion protein for use according to any one of claims 1-27, whereinthe IL13 comprises an amino acid sequence with between 1 and 10 aminoacid deletions, insertions, substitutions, or a combination thereofrelative to a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs: 9-15.
 31. The fusion protein for useaccording to any one of claims 1-30, wherein the regulatory cytokinecomprises an amino acid sequence with at least 90% sequence identity toa sequence selected from the group consisting of SEQ ID NO: 1, any oneof SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO:29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ IDNo: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO:
 45. 32. The fusionprotein for use according to any one of claims 1-30, wherein theregulatory cytokine comprises an amino acid sequence that is selectedfrom the group consisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28,SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7,SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO:18, SEQ ID NO: 36, and SEQ ID NO:
 45. 33. The fusion protein for useaccording to any one of claims 1-30, wherein the regulatory cytokinecomprises an amino acid sequence with between 1 and 10 amino aciddeletions, insertions, substitutions, or a combination thereof relativeto a sequence selected from the group consisting SEQ ID NO: 1, any oneof SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO:29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ IDNo: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO:
 45. 34. The fusionprotein for use according to any one of claims 1-33, wherein the IL13and the regulatory cytokine are covalently linked.
 35. The fusionprotein for use according to any one of claims 1-34, wherein the IL13and the regulatory cytokine are joined by a linker.
 36. The fusionprotein for use according to any one of claims 1-35, wherein a Cterminus of the IL13 is joined to an N-terminus of the cytokine,optionally via a linker.
 37. The fusion protein for use according to anyone of claims 1-35, wherein an N terminus of the IL13 is joined to aC-terminus of the cytokine, optionally via a linker.
 38. The fusionprotein for use according to any one of claims 1-37, wherein the fusionprotein further comprises one or more chemical modifications.
 39. Thefusion protein for use according to any one of claims 1-38, wherein theone or more chemical modifications are selected from the groupconsisting of glycosylation, fucosylation, sialylation, and pegylation.40. The fusion protein for use according to any one of claims 1-39,wherein the protein construct comprises an affinity tag.
 41. The fusionprotein for use according to any one of claim 1 or 7-40, wherein theneuropathy is post-traumatic peripheral neuropathy, post-operativeperipheral neuropathy, diabetic peripheral neuropathy, inflammatoryperipheral neuropathy, HIV-associated neuropathy, chemotherapy-inducedneuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy,cranial neuropathy, predominantly motor neuropathy, predominantlysensory neuropathy, sensory-motor neuropathy, autonomic neuropathy,idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia,glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia,atypical trigeminal neuralgia, sciatica, brachial plexopathy, orintercostal neuralgia.
 42. The fusion protein for use according to anyone of claims 1 and 7-41, wherein the neuropathy is associated withpain, numbness, weakness, burning, atrophy, tingling, twitching, or acombination thereof.
 43. The fusion protein for use according to any oneof claims 2 and 7-40, wherein the pain is chronic pain.
 44. The fusionprotein for use according to any one of claims 2, 7-40, and 43, whereinthe pain is pathological pain, inflammatory pain, neuropathic pain,nociceptive pain, or mixed nociceptive-neuropathic pain.
 45. The fusionprotein for use according to any one of claims 2, 7-40, and 43-44,wherein the pain is visceral nociceptive pain, non-visceral nociceptivepain, peripheral neuropathic pain, central neuropathic pain, or acombination thereof.
 46. The fusion protein for use according to any oneof claims 2, 7-40, and 43-45, wherein the pain is post-operativeorthopedic surgery pain, musculoskeletal pain, chemotherapy-associatedpain, chemotherapy-induced allodynia, post-spinal cord injury pain,post-stroke pain, low back pain, cancer pain, or chronic visceral pain.47. The fusion protein for use according to any one of claims 2, 7-40,and 43-45, wherein the pain is associated with irritable bowel syndrome,inflammatory bowel disease, rheumatoid arthritis, ankylosingspondylitis, post-herpetic neuralgia, trigeminal neuralgia,post-traumatic peripheral neuropathy, post-operative peripheralneuropathy, diabetic peripheral neuropathy, inflammatory peripheralneuropathy, HIV-associated neuropathy, peripheral neuropathy, nerveentrapment syndrome, chemotherapy-induced neuropathy, multiplesclerosis, chemotherapy-induced neurodegeneration, complex regional painsyndrome, osteoarthritis, fibromyalgia, polymyalgia rheumatica,myofascial pain syndrome, Alzheimer's disease, Parkinson's disease,Huntington's disease, polyneuropathy, or amyotrophic lateral sclerosis.48. The fusion protein for use according to any one of claims 2, 7-40,and 43-45, wherein the pain is associated with Alpers' Disease,Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, AutoimmuneAtrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular Necrosis,Back Pain, Batten Disease, Behçet's Disease (Syndrome), BreakthroughPain, Burning Mouth Syndrome, Bursitis, Central Autosomal DominantArteriopathy with Subcortical Infarcts and Leukoencephalopathy(Cadasil), Cerebral ischemia, Cerebro-Oculo-Facio-Skeletal Syndrome(COFS), Carpal Tunnel syndrome, Cauda Equina Syndrome, Central PainSyndrome, Cerebral Palsy, Cerebrospinal Fluid (CSF) Leaks, CervicalStenosis, Charcot-Marie-Tooth (CMT) Disease, Chronic FunctionalAbdominal Pain (CFAP), Chronic Pancreatitis, Collapsed Lung(Pneumothorax), Corticobasal Degeneration, Compression injury, CornealNeuropathic Pain, Crush syndrome, Degenerative Disc Disease,Dermatomyositis, Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS),Endometriosis, Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia,Failed Back Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia,Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains,Herniated disc, Hydrocephalus, Intercostal Neuraligia, InterstitialCystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy BodyDementia, Loin Pain-Haematuria Syndrome, Lyme Disease, MeralgiaParesthetica, Mitochondrial Disorders, Mixed dementia, Motor neuronediseases (MND), Monomelic Amyotrophy, Multiple system atrophy (MSA),Myositis, Neck Pain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis,Paget's Disease, Parsonage Turner Syndrome, Pelvic Pain, PeripheralNeuropathy, Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis,Polymyalgia Rhuematica, Polymyositis, Post Herniorraphy Pain Syndrome,Post Mastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy PainSyndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, PsoriaticArthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome,Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis,Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), Herpes Zoster Shingles, SpasmodicTorticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, SpinalStenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, ThoracicOutlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, TraumaticBrain Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combinationthereof.
 49. The fusion protein for use according to any one of claims 3and 7-40, wherein the neurodegeneration or neuroinflammation comprisesAlzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, multiple sclerosis, spinocerebellarataxia, or spinal muscular atrophy.
 50. The fusion protein for useaccording to any one of claims 4 and 7-40, wherein the inflammationcomprises chronic inflammation.
 51. The fusion protein for use accordingto any one of claims 4, 7-40, and 50, wherein the inflammation compriseslocal inflammation or systemic inflammation.
 52. The fusion protein foruse according to any one of claims 4, 7-40, and 50-51, wherein theinflammation is associated with inflammatory bowel disease, irritablebowel syndrome, osteoarthritis, rheumatoid arthritis,glomerulonephritis, sepsis, adult respiratory distress syndrome,dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosingspondylarthritis, systemic lupus erythematosus, vasculitis, gout,allotransplantation, xenotransplantation, an autoimmune disease,Sjogren's disease, a burn injury, trauma, stroke, myocardial infarction,atherosclerosis, diabetes mellitus, extracorporeal dialysis and bloodoxygenation, ischemia-reperfusion injuries, and toxicity induced by thein vivo administration of cytokines or other therapeutic monoclonalantibodies.
 53. The fusion protein for use according to any one ofclaims 1-5 and 7-52, wherein nerve fiber degeneration is reduced. 54.The fusion protein for use according to any one of claims 1-5 and 7-53,wherein nerve fiber loss is reduced.
 55. The fusion protein for useaccording to any one of claims 1-5 and 7-54, wherein maintenance ofnerve fiber density is promoted.
 56. The fusion protein for useaccording to any one of claims 1-5 and 7-55, wherein nerve fiberregrowth is promoted.
 57. The fusion protein for use according to anyone of claims 1-5 and 7-56 wherein neuroprotection in the centralnervous system is promoted.
 58. The fusion protein for use according toany one of claims 1-5 and 7-57, wherein neuroprotection in theperipheral nervous system is promoted.
 59. The fusion protein for useaccording to any one of claims 1-55 and 7-58, wherein intraepidermalnerve fiber loss is reduced.
 60. The fusion protein for use according toany one of claims 1-59, wherein the neuronal dysfunction is reduced. 61.The fusion protein for use according to any one of claims 1-5 and 7-60,wherein the fusion protein elicits a therapeutic effect of greatermagnitude than equivalent amounts of the IL13, the regulatory cytokine,or a combination thereof.
 62. The fusion protein for use according toany one of claims 1-5 and 7-61, wherein the fusion protein elicits atherapeutic effect of greater duration than equivalent amounts of theIL13, the regulatory cytokine, or a combination thereof.
 63. The fusionprotein for use according to any one of claims 1-62, wherein the fusionprotein is present in a pharmaceutical composition comprising the fusionprotein and one or more pharmaceutically-acceptable excipients.
 64. Thefusion protein for use according to claim 63, wherein the pharmaceuticalcomposition is in a unit dosage form.
 65. The fusion protein for useaccording to any one of claims 63-64, wherein the fusion protein ispresent in the pharmaceutical composition at a concentration of about 50μg per mL to about 100 mg per mL.
 66. The fusion protein for useaccording to any one of claims 63-65, wherein the fusion protein isformulated for administration in a dose of between about 0.5 μg per kgof body weight to about 1 mg per kg of body weight.
 67. The fusionprotein for use according to any one of claims 63-66, wherein the fusionprotein is formulated for administration in a controlled releaseformulation.
 68. The fusion protein for use according to any one ofclaims 63-67, wherein the fusion protein is formulated foradministration by a parenteral, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intrasternal, intracerebral, intraocular, intralesional,intracerebroventricular, intracisternal, or intraparenchymal route. 69.The fusion protein for use according to any one of claims 6-40, whereinthe nervous system cell is a neuron.
 70. The fusion protein for useaccording to any one of claims 6-40, wherein the nervous system cell isa central nervous system cell.
 71. The fusion protein for use accordingto any one of claims 6-40, wherein the nervous system cell is aperipheral nervous system cell.
 72. The fusion protein for use accordingclaim 69, wherein the neuron is a sensory neuron.
 73. The fusion proteinfor use according to claim 69, wherein the neuron is a somatosensoryneuron.
 74. The fusion protein for use according to claim 69, whereinthe neuron is a visceral sensory neuron.
 75. The fusion protein for useaccording to claim 69, wherein the neuron is a nociceptor.
 76. Thefusion protein for use according to claim 69, wherein the neuron is anautonomic neuron.
 77. The fusion protein for use according to any one ofclaims 6-40 and 70-71, wherein the nervous system cell is a glial cell.78. The fusion protein for use according to any one of claims 6-40 and70-71, wherein the nervous system cell is a microglial cell.
 79. Thefusion protein for use according to any one of claims 6-40 and 70-71,wherein the nervous system cell is an infiltrating cell.
 80. The fusionprotein for use according to any one of claims 6-40 and 70-71, whereinthe nervous system cell is an infiltrating macrophage.
 81. The fusionprotein for use according to any one of claims 6-40 and 69-81, whereinthe signaling pathway is modulated in a presence of a pro-inflammatorymediator.
 82. A method of treating neuropathy in a subject in needthereof, comprising administering to the subject an effective amount ofa fusion protein that comprises an interleukin 13 (IL13) amino acidsequence and a regulatory cytokine amino acid sequence.
 83. A method oftreating pain in a subject in need thereof, comprising administering tothe subject an effective amount of a fusion protein that comprises aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence.
 84. A method of treating neurodegeneration orneuroinflammation in a subject in need thereof, comprising administeringto the subject an effective amount of a fusion protein that comprises aninterleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence.
 85. A method of treating inflammation in a subjectin need thereof, comprising administering to the subject an effectiveamount of a fusion protein that comprises an interleukin 13 (IL13) aminoacid sequence and a regulatory cytokine amino acid sequence.
 86. Amethod of promoting neuroprotection in a subject in need thereof,comprising administering to the subject an effective amount of a fusionprotein that comprises an interleukin 13 (IL13) amino acid sequence anda regulatory cytokine amino acid sequence.
 87. A method of modulatingactivity of a signaling pathway in nervous system cell, comprisingcontacting the nervous system cell with a fusion protein that comprisesan interleukin 13 (IL13) amino acid sequence and a regulatory cytokineamino acid sequence.
 88. The method of any one of claims 82-87, whereinthe regulatory cytokine is selected from the group consisting of aninterleukin 4 (IL4), an interleukin 10 (IL10), an interleukin 33 (IL33),a transforming growth factor beta 1 (TGFβ1), a transforming growthfactor beta 2 (TGFβ2), and an additional interleukin 13 (aIL13).
 89. Themethod of any one of claims 82-88, wherein the regulatory cytokine isIL4.
 90. The method of any one of claims 82-88, wherein the regulatorycytokine is IL10.
 91. The method of any one of claims 82-88, wherein theregulatory cytokine is IL33.
 92. The method of any one of claims 82-88,wherein the regulatory cytokine is an interleukin 27 (IL27).
 93. Themethod of any one of claims 82-88, wherein the regulatory cytokine isTGFβ1.
 94. The method of any one of claims 82-88, wherein the regulatorycytokine is TGFβ2.
 95. The method of any one of claims 82-88, whereinthe regulatory cytokine is an aIL13.
 96. The method of any one of claims82-88, wherein the IL13 comprises a wild type IL13.
 97. The method ofany one of claims 82-88, wherein the IL13 is a mammalian IL13.
 98. Themethod of any one of claims 82-88, wherein the IL13 is a human IL13. 99.The method of any one of claims 82-98, wherein the regulatory cytokinecomprises a wild type regulatory cytokine.
 100. The method of any one ofclaims 82-99, wherein the regulatory cytokine is a mammalian regulatorycytokine.
 101. The method of any one of claims 82-99, wherein theregulatory cytokine is a human regulatory cytokine.
 102. The method ofany one of claims 82-87 and 96-101, wherein the interleukin 27 comprisesan interleukin 27 alpha (IL27A).
 103. The method of any one of claims82-87 and 96-101, wherein the IL27A comprises an L134C substitutionrelative to SEQ ID NO:
 36. 104. The method of any one of claims 82-103,wherein the IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1)with an affinity that is less than two fold increased and less than twofold decreased compared to a wild type IL13.
 105. The method of any oneof claims 82-104, wherein the IL13 binds to interleukin 13 receptoralpha 2 (IL-13Rα2) with an affinity that is less than two fold increasedand less than two fold decreased compared to a wild type IL13.
 106. Themethod of any one of claims 82-105, wherein the IL13 binds to aninterleukin 4 receptor alpha (IL-4Rα) with an affinity that is less thantwo fold increased and less than two fold decreased compared to a wildtype IL13.
 107. The method of any one of claims 82-98 and 100-106,wherein the regulatory cytokine amino acid sequence is a derivativesequence that binds to all subunits of a receptor of the regulatorycytokine with a comparable affinity as a wild type regulatory cytokine.108. The method of any one of claims 82-98 and 100-107, wherein theregulatory cytokine amino acid sequence is a derivative sequence thatactivates a native receptor of the regulatory cytokine.
 109. The methodof any one of claims 82-95 and 97-108, wherein the IL13 comprises anamino acid sequence with at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 2 and any one of SEQ IDNOs: 9-15.
 110. The method of any one of claims 82-109, wherein the IL13comprises an amino acid sequence that is selected from the groupconsisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15.
 111. Themethod of any one of claims 82-95 and 97-109, wherein the IL13 comprisesan amino acid sequence with between 1 and 10 amino acid deletions,insertions, substitutions, or a combination thereof relative to asequence selected from the group consisting of SEQ ID NO: 2 and any oneof SEQ ID NOs: 9-15.
 112. The method of any one of claims 82-111,wherein the regulatory cytokine comprises an amino acid sequence with atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ ID NO: 5,SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ ID NO: 21,SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQ ID NO:36, and SEQ ID NO:
 45. 113. The method of any one of claims 82-106 and109-112, wherein the regulatory cytokine comprises an amino acidsequence that is selected from the group consisting of SEQ ID NO: 1, anyone of SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ IDNO: 29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQID No: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO:
 45. 114. Themethod of any one of claims 82-106 and 109-113, wherein the regulatorycytokine comprises an amino acid sequence with between 1 and 10 aminoacid deletions, insertions, substitutions, or a combination thereofrelative to a sequence selected from the group consisting SEQ ID NO: 1,any one of SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQID NO: 29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22,SEQ ID No: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO:
 45. 115. Themethod of any one of claims 82-114, wherein the IL13 and the regulatorycytokine are covalently linked.
 116. The method of any one of claims82-115, wherein the IL13 and the regulatory cytokine are joined by alinker.
 117. The method of any one of claims 82-115, wherein a Cterminus of the IL13 is joined to an N-terminus of the cytokine,optionally via a linker.
 118. The method of any one of claims 82-115,wherein an N terminus of the IL13 is joined to a C-terminus of thecytokine, optionally via a linker.
 119. The method of any one of claims82-118, wherein the fusion protein further comprises one or morechemical modifications.
 120. The method of any one of claims 82-119,wherein the one or more chemical modifications are selected from thegroup consisting of glycosylation, fucosylation, sialylation, andpegylation.
 121. The method of any one of claims 82-120, wherein theprotein construct comprises an affinity tag.
 122. The method of any oneof claims 82 and 88-121, wherein the neuropathy is post-traumaticperipheral neuropathy, post-operative peripheral neuropathy, diabeticperipheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, chemotherapy-induced neuropathy,polyneuropathy, mononeuropathy, multiple mononeuropathy, cranialneuropathy, predominantly motor neuropathy, predominantly sensoryneuropathy, sensory-motor neuropathy, autonomic neuropathy, idiopathicneuropathy, post-herpetic neuralgia, trigeminal neuralgia,glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia,atypical trigeminal neuralgia, sciatica, brachial plexopathy, orintercostal neuralgia.
 123. The method of any one of claims 82 and88-122, wherein the neuropathy is associated with pain, numbness,weakness, burning, atrophy, tingling, twitching, or a combinationthereof.
 124. The method of any one of claims 83 and 88-121, wherein thepain is chronic pain.
 125. The method of any one of claims 83, 88-121and 124, wherein the pain is neuropathic pain, nociceptive pain, ormixed nociceptive-neuropathic pain.
 126. The method of any one of claims83, 88-121 and 124, wherein the pain is visceral nociceptive pain,non-visceral nociceptive pain, peripheral neuropathic pain, centralneuropathic pain, or a combination thereof.
 127. The method of any oneof claims 83, 88-121 and 124, wherein the pain is post-operativeorthopedic surgery pain, musculoskeletal pain, chemotherapy-associatedpain, chemotherapy-induced allodynia, post-spinal cord injury pain,post-stroke pain, low back pain, cancer pain, or chronic visceral pain.128. The method of any one of claims 83, 88-121 and 124-127, wherein thepain is associated with irritable bowel syndrome, inflammatory boweldisease, rheumatoid arthritis, ankylosing spondylitis, post-herpeticneuralgia, trigeminal neuralgia, post-traumatic peripheral neuropathy,post-operative peripheral neuropathy, diabetic peripheral neuropathy,inflammatory peripheral neuropathy, HIV-associated neuropathy,peripheral neuropathy, nerve entrapment syndrome, chemotherapy-inducedneuropathy, multiple sclerosis, chemotherapy-induced neurodegeneration,complex regional pain syndrome, osteoarthritis, fibromyalgia,polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease,Parkinson's disease, Huntington's disease, polyneuropathy, oramyotrophic lateral sclerosis.
 129. The method of any one of claims 83,88-121 and 124-127, wherein the pain is associated with Alpers' Disease,Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, AutoimmuneAtrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular Necrosis,Back Pain, Batten Disease, Behçet's Disease (Syndrome), BreakthroughPain, Burning Mouth Syndrome, Bursitis, Central Autosomal DominantArteriopathy with Subcortical Infarcts and Leukoencephalopathy(Cadasil), Cerebral ischemia, Cerebro-Oculo-Facio-Skeletal Syndrome(COFS), Carpal Tunnel syndrome, Cauda Equina Syndrome, Central PainSyndrome, Cerebral Palsy, Cerebrospinal Fluid (CSF) Leaks, CervicalStenosis, Charcot-Marie-Tooth (CMT) Disease, Chronic FunctionalAbdominal Pain (CFAP), Chronic Pancreatitis, Collapsed Lung(Pneumothorax), Corticobasal Degeneration, Compression injury, CornealNeuropathic Pain, Crush syndrome, Degenerative Disc Disease,Dermatomyositis, Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS),Endometriosis, Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia,Failed Back Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia,Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains,Herniated disc, Hydrocephalus, Intercostal Neuraligia, InterstitialCystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy BodyDementia, Loin Pain-Haematuria Syndrome, Lyme Disease, MeralgiaParesthetica, Mitochondrial Disorders, Mixed dementia, Motor neuronediseases (MND), Monomelic Amyotrophy, Multiple system atrophy (MSA),Myositis, Neck Pain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis,Paget's Disease, Parsonage Turner Syndrome, Pelvic Pain, PeripheralNeuropathy, Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis,Polymyalgia Rhuematica, Polymyositis, Post Herniorraphy Pain Syndrome,Post Mastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy PainSyndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, PsoriaticArthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome,Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis,Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), Herpes Zoster Shingles, SpasmodicTorticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, SpinalStenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, ThoracicOutlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, TraumaticBrain Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combinationthereof.
 130. The method of any one of claims 84 and 88-121, wherein theneurodegeneration or neuroinflammation comprises Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, multiple sclerosis, spinocerebellar ataxia, or spinalmuscular atrophy.
 131. The method of any one of claims 85 and 88-121,wherein the inflammation comprises chronic inflammation.
 132. The methodof any one of claims 85, 88-121 and 131, wherein the inflammationcomprises local inflammation or systemic inflammation.
 133. The methodof any one of claims 85, 88-121 and 131, wherein the inflammation isassociated with inflammatory bowel disease, irritable bowel syndrome,osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adultrespiratory distress syndrome, dermatitis, sarcoidosis, allergicinflammation, psoriasis, ankylosing spondylarthritis, systemic lupuserythematosus, vasculitis, gout, allotransplantation,xenotransplantation, an autoimmune disease, Sjogren's disease, a burninjury, trauma, stroke, myocardial infarction, atherosclerosis, diabetesmellitus, extracorporeal dialysis and blood oxygenation,ischemia-reperfusion injuries, and toxicity induced by the in vivoadministration of cytokines or other therapeutic monoclonal antibodies.134. The method of any one of claims 82-133, wherein IL13 treatment isindicated.
 135. The method of any one of claims 82-134, wherein IL4,IL10, IL27, IL33, TGFβ1, or TGFβ2 treatment is indicated.
 136. Themethod of any one of claims 82-86 and 88-135, wherein the method reducesnerve fiber degeneration.
 137. The method of any one of claims 82-86 and88-135, wherein the method reduces nerve fiber loss.
 138. The method ofany one of claims 82-86 and 88-135, wherein the method promotesmaintenance of nerve fiber density.
 139. The method of any one of claims82-86 and 88-135, wherein the method promotes nerve fiber regrowth. 140.The method of any one of claims 82-86 and 88-135, wherein the methodpromotes neuroprotection in the central nervous system.
 141. The methodof any one of claims 82-86 and 88-135, wherein the method promotesneuroprotection in the peripheral nervous system.
 142. The method of anyone of claims 82-86 and 88-135, wherein the method reducesintraepidermal nerve fiber loss.
 143. The method of any one of claims82-86 and 88-135, wherein the method reduces neuronal dysfunction. 144.The method of any one of claims 82-86 and 88-143, wherein administeringthe fusion protein elicits a therapeutic effect of greater magnitudethan administering equivalent amounts of the IL13, the regulatorycytokine, or a combination thereof.
 145. The method of any one of claims82-86 and 88-144, wherein administering the fusion protein elicits atherapeutic effect of greater duration than administering equivalentamounts of the IL13, the regulatory cytokine, or a combination thereof.146. The method of any one of claims 82-86 and 88-144, whereinadministering results in a higher magnitude of pain alleviation ascompared to a comparable amount of IL13 and the regulatory cytokineadministered individually or in combination as measured by mechanicalsensitivity to von Frey hairs in a paclitaxel-induced mouse model ofneuropathy.
 147. The method of any one of claims 82-146 wherein thefusion protein is present in a pharmaceutical composition comprising thefusion protein and one or more pharmaceutically-acceptable excipients.148. The method of claim 147, wherein the composition is in a unitdosage form.
 149. The method of any one of claims 147-148, wherein thefusion protein is present in the pharmaceutical composition at aconcentration of about 50 μg to about 100 mg per mL.
 150. The method ofany one of claims 82-86 and 88-149, wherein the fusion protein isadministered in a dose of between about 0.5 μg to 1 mg per kg of bodyweight.
 151. The method of any one of claims 82-86 and 88-150, whereinthe fusion protein is administered in a controlled release formulation.152. The method of any one of claims 82-86 and 88-151, wherein thefusion protein is administered by a parenteral, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural, intrasternal, intracerebral, intraocular, intralesional,intracerebroventricular, intracisternal, or intraparenchymal route. 153.The method of any one of claims 87-121, wherein the nervous system cellis a neuron.
 154. The method of any one of claims 87-121 and 153,wherein the nervous system cell is a central nervous system cell. 155.The method of any one of claims 87-121 and 153, wherein the nervoussystem cell is a peripheral nervous system cell.
 156. The method of anyone of claims 153-155, wherein the neuron is a sensory neuron.
 157. Themethod of any one of claims 153-155, wherein the neuron is asomatosensory neuron.
 158. The method of any one of claims 153-155,wherein the neuron is a visceral sensory neuron.
 159. The method of anyone of claims 153-155, wherein the neuron is a nociceptor.
 160. Themethod of any one of claims 153-155, wherein the neuron is an autonomicneuron.
 161. The method of any one of claims 87-121 and 154-155, whereinthe nervous system cell is a glial cell.
 162. The method of any one ofclaims 87-121 and 154-155, wherein the nervous system cell is amicroglial cell.
 163. The method of any one of claims 87-121 and154-155, wherein the nervous system cell is an infiltrating cell. 164.The method of any one of claims 87-121 and 154-155, wherein the nervoussystem cell is an infiltrating macrophage.
 165. The method of any one ofclaims 87-121 and 153-164, wherein the signaling pathway is modulated ina presence of a pro-inflammatory mediator.
 166. A fusion proteincomprising an interleukin 13 (IL13) amino acid sequence that is a wildtype IL13 sequence and a regulatory cytokine amino acid sequence.
 167. Afusion protein comprising an interleukin 13 (IL13) amino acid sequenceand a regulatory cytokine amino acid sequence that is a wild typesequence.
 168. A fusion protein comprising an interleukin 13 (IL13)amino acid sequence and a regulatory cytokine amino acid sequence,wherein the IL13 amino acid sequence is an IL13 derivative sequence thatbinds to interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13receptor alpha 2 (IL-13Rα2), and interleukin 4 receptor alpha (IL-4Rα)with a comparable affinity as a wild type interleukin 13 sequence. 169.A fusion protein comprising an interleukin 13 (IL13) amino acid sequenceand a regulatory cytokine amino acid sequence, wherein the regulatorycytokine amino acid sequence is a derivative sequence that binds to allreceptor subunits that a wild type version of the regulatory cytokinebinds with a comparable affinity as the wild type regulatory cytokine.170. The fusion protein of any one of claims 166-169, wherein theregulatory cytokine is selected from the group consisting of aninterleukin 4 (IL4), an interleukin 10 (IL10), an interleukin 33 (IL33),a transforming growth factor beta 1 (TGFβ1), a transforming growthfactor beta 2 (TGFβ2), and an additional interleukin 13 (aIL13). 171.The fusion protein of any one of claims 166-169, wherein the regulatorycytokine is IL4.
 172. The fusion protein of any one of claims 166-169,wherein the regulatory cytokine is IL10.
 173. The fusion protein of anyone of claims 166-169, wherein the regulatory cytokine is IL33.
 174. Thefusion protein of any one of claims 166-169, wherein the regulatorycytokine is an interleukin 27 (IL27).
 175. The fusion protein of any oneof claims 166-169, wherein the regulatory cytokine is TGFβ1.
 176. Thefusion protein of any one of claims 166-169, wherein the regulatorycytokine is TGFβ2.
 177. The fusion protein of any one of claims 166-169,Wherein the regulatory cytokine is an additional interleukin 13 (aIL13).178. The fusion protein of any one of claims 166-177, wherein the IL13is a mammalian IL13.
 179. The fusion protein of any one of claims166-178, wherein the IL13 is a human IL13.
 180. The fusion protein ofany one of claims 167 and 169-179, wherein the IL13 comprises a wildtype IL13.
 181. The fusion protein of any one of claims 166, 168, and170-180 wherein the regulatory cytokine comprises a wild type regulatorycytokine.
 182. The fusion protein of any one of claims 166-181, whereinthe regulatory cytokine is a mammalian regulatory cytokine.
 183. Thefusion protein of any one of claims 166-182, wherein the regulatorycytokine is a human regulatory cytokine.
 184. The fusion protein of anyone of claims 174 and 178-183, wherein the interleukin 27 comprises aninterleukin 27 alpha (IL27A).
 185. The fusion protein of any one ofclaims 174 and 178-184, wherein the IL27A comprises an L134Csubstitution relative to SEQ ID NO:
 36. 186. The fusion protein of anyone of claims 167, 169-179, and 181-185, wherein the IL13 binds tointerleukin 13 receptor alpha 1 (IL-13Rα1) with an affinity that is lessthan two fold increased and less than two fold decreased compared to awild type IL13.
 187. The fusion protein of any one of claims 167,169-179, and 181-186, wherein the IL13 binds to interleukin 13 receptoralpha 2 (IL-13Rα2) with an affinity that is less than two fold increasedand less than two fold decreased compared to a wild type IL13.
 188. Thefusion protein of any one of claims 167, 169-179, and 181-187, whereinthe IL13 binds to an interleukin 4 receptor alpha (IL-4Rα) with anaffinity that is less than two fold increased and less than two folddecreased compared to a wild type IL13.
 189. The fusion protein of anyone of claims 166, 168, 170-180, and 182-188, wherein the regulatorycytokine amino acid sequence is a derivative sequence that binds to allsubunits of a receptor of the regulatory cytokine with a comparableaffinity as a wild type regulatory cytokine.
 190. The fusion protein ofany one of claims 166, 168, 170-180, and 182-189, wherein the regulatorycytokine amino acid sequence is a derivative sequence that activates anative receptor of the regulatory cytokine.
 191. The fusion protein ofany one of claims 166-190, wherein the IL13 comprises an amino acidsequence with at least 90% sequence identity to a sequence selected fromthe group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15.192. The fusion protein of any one of claims 166, 167, and 169-191,wherein the IL13 comprises an amino acid sequence that is selected fromthe group consisting of SEQ ID NO: 2 and any one of SEQ ID NOs: 9-15.193. The fusion protein of any one of claims 167-179 and 181-191,wherein the IL13 comprises an amino acid sequence with between 1 and 10amino acid deletions, insertions, substitutions, or a combinationthereof relative to a sequence selected from the group consisting of SEQID NO: 2 and any one of SEQ ID NOs: 9-15.
 194. The fusion protein of anyone of claims 166-193, wherein the regulatory cytokine comprises anamino acid sequence with at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 1, any one of SEQ IDNOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQID NO: 18, SEQ ID NO: 36, and SEQ ID NO:
 45. 195. The fusion protein ofany one of claims 166-168, 170-188, and 191-194, wherein the regulatorycytokine comprises an amino acid sequence that is selected from thegroup consisting of SEQ ID NO: 1, any one of SEQ ID NOs: 26-28, SEQ IDNO: 5, SEQ ID NO: 6, any one of SEQ ID NO: 29-34, SEQ ID NO: 7, SEQ IDNO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID No: 35, SEQ ID NO: 18, SEQID NO: 36, and SEQ ID NO:
 45. 196. The fusion protein of any one ofclaims 166, 168-180, and 182-194, wherein the regulatory cytokinecomprises an amino acid sequence with between 1 and 10 amino aciddeletions, insertions, substitutions, or a combination thereof relativeto a sequence selected from the group consisting SEQ ID NO: 1, any oneof SEQ ID NOs: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, any one of SEQ ID NO:29-34, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 8, SEQ ID NO: 22, SEQ IDNO: 35, SEQ ID NO: 18, SEQ ID NO: 36, and SEQ ID NO:
 45. 197. The fusionprotein of any one of claims 166-196, wherein the IL13 and theregulatory cytokine are covalently linked.
 198. The fusion protein ofany one of claims 166-197, wherein the IL13 and the regulatory cytokineare joined by a linker.
 199. The fusion protein of any one of claims166-198, wherein a C terminus of the IL13 is joined to an N-terminus ofthe cytokine, optionally via a linker.
 200. The fusion protein of anyone of claims 166-198, wherein an N terminus of the IL13 is joined to aC-terminus of the cytokine, optionally via a linker.
 201. The fusionprotein of any one of claims 166-200, wherein the fusion protein furthercomprises one or more chemical modifications.
 202. The fusion protein ofclaim 201, wherein the one or more chemical modifications are selectedfrom the group consisting of glycosylation, fucosylation, sialylation,and pegylation.
 203. The fusion protein of any one of claims 166-202,wherein the protein construct comprises an affinity tag.
 204. A nucleicacid molecule comprising a polynucleotide sequence that encodes thefusion protein of any one of claims 166-203.
 205. The nucleic acidmolecule of claim 204, wherein the polynucleotide is codon optimized forexpression in the cell.
 206. A nucleic acid vector comprising thepolynucleotide sequence of claim 204 or claim
 205. 207. A cellcomprising the nucleic acid of claim 204 or claim
 205. 208. Apharmaceutical composition comprising the fusion protein of any one ofclaims 166-203 and a pharmaceutically-acceptable excipient.
 209. Apharmaceutical composition comprising the nucleic acid vector of claim206 and a pharmaceutically-acceptable excipient.
 210. The pharmaceuticalcomposition of claim 208 or claim 209, wherein the pharmaceuticalcomposition is in a unit dosage form.
 211. The pharmaceuticalcomposition of any one of claims 208-210, wherein the fusion protein ispresent in the pharmaceutical composition at about 50 μg to about 100 mgper mL.
 212. The pharmaceutical composition of any one of claims208-211, wherein the fusion protein is formulated for administration asa dose of between about 0.5 μg to 1 mg per kg of body weight.
 213. Thepharmaceutical composition of any one of claims 208-212, wherein thefusion protein formulated for administration as a controlled releaseformulation.
 214. The pharmaceutical composition of any one of claims208-213, wherein the pharmaceutical composition is formulated foradministration by a parenteral, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intrasternal, intracerebral, intraocular, intralesional,intracerebroventricular, intracisternal, or intraparenchymal route. 215.A method of treating a condition in a subject in need thereof,comprising administering to the subject an effective amount of thepharmaceutical composition of any one of claims 208-214.
 216. A methodof producing a fusion protein, comprising culturing a cell underconditions that permit the production of the fusion protein, wherein thecell comprises the polynucleotide sequence of claim 204 or claim 205.217. The method of claim 216, further comprising harvesting the fusionprotein.
 218. The method of claim 216, further comprising purifying thefusion protein from harvested culture medium.
 219. A fusion proteincomprising an interleukin 13 and an interleukin chosen from interleukin4, interleukin 10, interleukin 33, transforming growth factor beta 1,transforming growth factor beta 2, and interleukin
 13. 220. A fusionprotein according to claim 219, wherein said interleukin 13 and saidinterleukin chosen from interleukin 4, interleukin 10, interleukin 33,transforming growth factor beta 1, transforming growth factor beta 2,and interleukin 13, are linked by a linker sequence.
 221. A fusionprotein according to any one of claims 219-220, wherein the interleukin13 is fused N-terminal of the interleukin chosen from interleukin 4,interleukin 10, interleukin 33, transforming growth factor beta 1,transforming growth factor beta 2, and interleukin
 13. 222. A fusionprotein according to any one of claims 219-220, wherein the interleukinchosen from interleukin 4, interleukin 10, interleukin 33, transforminggrowth factor beta 1, transforming growth factor beta 2, and interleukin13, is fused N-terminal of the interleukin
 13. 223. A fusion proteinaccording to any one of claims 219-222, which further comprises one ormore chemical modification(s).
 224. A fusion protein according to claim223, wherein the chemical modification is selected from the groupconsisting of glycosylation, fucosylation, sialylation, and pegylation.225. A fusion protein according to any one of claims 219-224, whereinsaid interleukin 13 is human interleukin
 13. 226. A fusion proteinaccording to any one of claims 219-225, wherein said interleukin 4 ishuman interleukin 4, and/or said interleukin 10 is human interleukin 10,and/or said interleukin 33 is human interleukin 33, and/or saidtransforming growth factor beta 1 is human transforming growth factorbeta 1, and/or said transforming growth factor beta 2 is humantransforming growth factor beta
 2. 227. A nucleic acid moleculecomprising a polynucleotide encoding the fusion protein according to oneof claims 219-226.
 228. A vector comprising the nucleic acid molecule ofclaim
 227. 229. A host cell comprising the nucleic acid moleculeaccording to claim 227 or the vector according to claim
 228. 230. Methodfor producing a fusion protein according to any one of claims 219-226,said method comprising the steps of: culturing a host cell according toclaim 229 under conditions permitting the production of the fusionprotein according to any one of claims 219-226; optionally, purifyingthe fusion protein from the conditioned culture medium.
 231. Apharmaceutical composition comprising the fusion protein according toany one of claims 219-226, and a pharmaceutically acceptable carrier.232. A fusion protein according to any one of claims 219-226 for use asa medicament.
 233. A fusion protein according to any one of claims219-226 for use in the prevention or treatment of a conditioncharacterized by chronic pain, neuro-inflammation or neuro-degeneration.234. A fusion protein for use according to claim 233, wherein saidcondition is further characterized by visceral or non-visceralnociceptive pain, peripheral or central neuropathic pain, or mixednociceptive-neuropathic pain, neuro-inflammation, and/orneuro-degeneration.
 235. A fusion protein for use according to claims233 or claim 234, wherein said condition is selected from the groupconsisting of post-operative orthopedic surgery pain, musculoskeletalpain, irritable bowel syndrome, inflammatory bowel disease, rheumatoidarthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminalneuralgia, post-traumatic or post-operative peripheral neuropathy,diabetic peripheral neuropathy, inflammatory peripheral neuropathy,HIV-associated neuropathy, painful peripheral neuropathy, nerveentrapment syndrome, chemotherapy-associated pain, chemotherapy-inducedallodynia, complex regional pain syndrome, post-spinal injury pain,post-stroke pain, multiple sclerosis, low back pain, osteoarthritis,cancer pain, chronic visceral pain, fibromyalgia, polymyalgiarheumatica, myofascial pain syndrome, Alzheimer's disease andParkinson's disease, Huntington's disease, and/or amyotrophic lateralsclerosis, or multiple sclerosis.
 236. A fusion protein according to anyone of claims 219-226 for use in the prevention or treatment of aclinical condition in a mammal, such as a human, for which interleukin13 is indicated.
 237. A fusion protein according to any one of claims219-226 for use in the prevention or treatment of a clinical conditionin a mammal, such as a human, for which interleukin 4 and/or interleukin10 and/or interleukin 33 and/or interleukin 27 and/or transforminggrowth factor beta 1 and/or transforming growth factor beta 2, isindicated.
 238. A gene therapy vector containing nucleotide sequence(s)coding for interleukin 13 and an interleukin chosen from interleukin 4,interleukin 10, interleukin 27, interleukin 33, transforming growthfactor beta 1, transforming growth factor beta 2, and interleukin 13,for use in the prevention or treatment of a condition characterized bychronic pain, neuro-inflammation and/or neuro-degeneration.
 239. A genetherapy vector according to claim 239, wherein said condition is furthercharacterized by visceral or non-visceral nociceptive pain, peripheralor central neuropathic pain, or mixed nociceptive-neuropathic pain,neuro-inflammation, and/or neuro-degeneration.
 240. A gene therapyvector according to claim 238 or claim 239, wherein said condition isselected from the group consisting of post-operative orthopedic surgerypain, musculoskeletal pain, irritable bowel syndrome, inflammatory boweldisease, rheumatoid arthritis, ankylosing spondylitis, post-herpeticneuralgia, trigeminal neuralgia, post-traumatic or post-operativeperipheral neuropathy, diabetic peripheral neuropathy, inflammatoryperipheral neuropathy, HIV-associated neuropathy, painful peripheralneuropathy, nerve entrapment syndrome, chemotherapy-associated pain,chemotherapy-induced allodynia, complex regional pain syndrome,post-spinal injury pain, post-stroke pain, multiple sclerosis, low backpain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia,polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's diseaseand Parkinson's disease, Huntington's disease, and/or amyotrophiclateral sclerosis, or multiple sclerosis.
 241. The fusion protein of anyone of claims 166-203 for use in crosslinking an interleukin 13 receptorwith a regulatory cytokine receptor.